Single-vector type I vectors

ABSTRACT

The invention relates to the production and use of Cas-encoding sequences and vectors comprising these. Aspects of the invention provide products, vectors, delivery vehicles, uses and methods for producing Cas-encoding sequences in bacterial or archaeal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Great Britain Patent Application No.1816700.7, filed Oct. 14, 2018, and Great Britain Patent Application No.1817509.1, filed Oct. 27, 2018, the contents of each of which are herebyincorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 786212000600SEQLIST.TXT,date recorded: Nov. 26, 2018, size: 6,008 bytes).

TECHNICAL FIELD

The invention relates to the production and use of Cas-encodingsequences and vectors comprising these. Aspects of the invention provideproducts, vectors, delivery vehicles, uses and methods for producingCas-encoding sequences in bacterial or archaeal cells.

BACKGROUND

The state of the art describes vectors and uses of these that employCRISPR/Cas systems. For example, reference is made to WO2017/118598,US20180140698, US20170246221, US20180273940, US20160115488,US20180179547, US20170175142, US20160024510, US20150064138,US20170022499, US20160345578, US20180155729, US20180200342,WO2017112620, WO2018081502, PCT/EP2018/066954, PCT/EP2018/066980,PCT/EP2018/071454 and U.S. Ser. No. 15/985,658 and equivalentpublications by the US Patent and Trademark Office (USPTO) or WIPO, thedisclosures of which are incorporated herein by reference.

SUMMARY OF THE INVENTION

The invention provides the following configurations.

In a First Configuration

A nucleic acid vector for introduction into a host cell, the vectorcomprising a first nucleotide sequence encoding a Type I Cas3 and asecond nucleotide sequence encoding one or more Cascade proteins,wherein the first and second sequences are under the control of one ormore promoters comprised by the vector for expression of the proteins inthe cell.

In an example, the vector comprises an operon for expression in the cellof the Cas3 and Cascade proteins from a Cas module, the modulecomprising the nucleotide sequences encoding the Cas3 and Cascadeproteins, and the operon comprising the Cas module under the control ofa promoter for controlling the expression of both the Cas3 and Cascadeproteins.

The invention also provides a delivery vehicle comprising the vector, aswell as a pharmaceutical composition comprising the vector or vehicleand a pharmaceutically acceptable diluent, excipient or carrier.

The invention also provides a method of treating or reducing the risk ofa disease or condition in a human or animal subject, the methodcomprising administering the vector, vehicle or composition to thesubject, and introducing the vector into target host bacterial orarchaeal cells in the subject (eg, in a gut microbiota, lung, eye orblood of the subject), wherein the Cas cuts (or otherwise modifies) oneor more target sequences in the target cells and the cells are killed orgrowth or proliferation of the cells is reduced.

In a Second Configuration

A method of amplifying copies of a DNA encoding a functional Cas protein(optionally a Cas nuclease) in a bacterial or archaeal production strainof cells, the method comprising

-   -   (a) Providing production strain cells, each cell comprising a        copy of said DNA, wherein each DNA comprises a nucleotide        sequence encoding said Cas, wherein the nucleotide sequence is        under the control of a promoter for controlling the expression        of the Cas in the production strain cell, the DNA comprising an        origin of replication that is operable in the cell for        replication of the DNA;    -   (b) Culturing the cells to allow replication of the DNA, whereby        the DNA is amplified; and    -   (c) Optionally isolating copies of the DNA,    -   Optionally wherein the promoter is an attenuated constitutive        promoter.

In a Third Configuration

Use of an attenuated promoter in a DNA construct comprising a nucleotidesequence encoding a functional Cas protein (optionally a Cas nuclease)that is under the control of the promoter, in a method of amplifyingcopies of the DNA in a population of bacterial or archaeal productionstrain cells, the method comprising culturing the cells to allowreplication of the DNA thereby amplifying the DNA in the cells, forenhancing the yield of amplified DNA produced by the production hostcells.

In a Fourth Configuration

Use of an attenuated promoter in a DNA construct comprising a nucleotidesequence encoding a functional Cas protein (optionally a Cas nuclease)that is under the control of the promoter, in a method of amplifyingcopies of the DNA in a population of bacterial or archaeal productionstrain cells, the method comprising culturing the cells to allowreplication of the DNA thereby amplifying the DNA in the cells, forreducing toxicity of the Cas in the production strain.

In a Fifth Configuration

Use of an attenuated promoter in a DNA construct comprising a nucleotidesequence encoding a functional Cas protein (optionally a Cas nuclease)that is under the control of the promoter, in a method of amplifyingcopies of the DNA in a population of bacterial or archaeal productionstrain cells, the method comprising culturing the cells to allowreplication of the DNA thereby amplifying the DNA in the cells, forreducing mutation of the DNA (optionally the Cas-encoding sequence) inthe production strain.

In a Sixth Configuration

Use of an attenuated promoter in a DNA construct comprising a nucleotidesequence encoding a functional Cas protein (optionally a Cas nuclease)that is under the control of the promoter, in a method of amplifyingcopies of the DNA in a population of bacterial or archaeal productionstrain cells, the method comprising culturing the cells to allowreplication of the DNA thereby amplifying the DNA in the cells, forpromoting production cell viability during the amplification of the DNA.

In a Seventh Configuration

Use of an attenuated promoter in a DNA construct comprising a nucleotidesequence encoding a functional Cas protein (optionally a Cas nuclease)that is under the control of the promoter, in a method of amplifyingcopies of the DNA in a population of bacterial or archaeal productionstrain cells, the method comprising culturing the cells to allowreplication of the DNA thereby amplifying the DNA in the cells, forreducing the occurrence of Cas cutting of DNA.

In an Eighth Configuration

A method for enhancing the yield of amplified copies of a DNA constructin a population of bacterial or archaeal production strain cells,wherein the construct comprises a nucleotide sequence encoding afunctional Cas protein (optionally a Cas nuclease) that is under thecontrol of a promoter, the method comprising culturing the cells toallow replication of the DNA thereby amplifying the DNA in the cells,wherein the promoter is an attenuated promoter.

In a Ninth Configuration

A method for reducing toxicity of a functional Cas protein (optionally aCas nuclease) in a population of bacterial or archaeal production straincells in a process of amplifying copies of a DNA construct, wherein theconstruct comprises a nucleotide sequence encoding the Cas and thesequence is under the control of a promoter, the method comprisingculturing the cells to allow replication of the DNA thereby amplifyingthe DNA in the cells, wherein the promoter is an attenuated promoter.

In a Tenth Configuration

A method for reducing mutation of a DNA construct encoding a functionalCas protein (optionally a Cas nuclease) in a population of bacterial orarchaeal production strain cells in a process of amplifying copies ofthe construct, wherein the construct comprises a nucleotide sequenceencoding the Cas and the sequence is under the control of a promoter,the method comprising culturing the cells to allow replication of theDNA thereby amplifying the DNA in the cells, wherein the promoter is anattenuated promoter.

In an Eleventh Configuration

A method for promoting production cell viability of a population ofbacterial or archaeal production strain cells in a process of amplifyingcopies of a DNA construct comprised by the cells, wherein the constructcomprises a nucleotide sequence encoding a functional Cas protein(optionally a Cas nuclease) and the sequence is under the control of apromoter, the method comprising culturing the cells to allow replicationof the DNA thereby amplifying the DNA in the cells, wherein the promoteris an attenuated promoter.

In a Twelfth Configuration

A method for reducing the occurrence of Cas nuclease cutting of a DNAconstruct in a population of bacterial or archaeal production straincells in a process of amplifying copies of the construct, wherein theconstruct comprises a nucleotide sequence encoding the Cas and thesequence is under the control of a promoter, the method comprisingculturing the cells to allow replication of the DNA thereby amplifyingthe DNA in the cells, wherein the promoter is an attenuated promoter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Type I CRISPR-Cas system of C. difficile targeting E. coliMG1655. (FIG. 1A) Layout of the CRISPR Guided Vector™, CGV™. Plasmid 1:pSC101 ori, pBAD promoter (induced by arabinose), cas3 and cascadegenes. Plasmid 2: pCloDF13 ori, pTac promoter (induced by IPTG), CRISPRarray. (FIG. 1B) Dilution series (10¹-10⁶) of drop spots (5 μl) of E.coli MG1655 harboring the CGV on LB agar plates with and withoutinducers. (FIG. 1C) CRISPR induction killed 99.9% of the population(grey bar). Growth in absence of induction is shown in black. CGV™refers to a CRISPR Guided Vector™, which is a nucleic acid vectorcomprising nucleotide sequences encoding CRISPR/Cas components.

FIGS. 2A-2C. Type I CRISPR-Cas system of C. difficile targeting E. coliMG1655. (FIG. 2A) Layout of the CRISPR Guided Vector™, CGV™. pSC101 ori,pTac promoter (induced by IPTG), CRISPR array, pBAD promoter (induced byarabinose), cas3 and cascade genes. (FIG. 2B) Dilution series (10¹-10⁶)of drop spots (5 μl) of E. coli MG1655 harboring the CGV on SM agarplates with and without inducers. (FIG. 2C) CRISPR induction killed 99%of the population (grey bar). Growth in absence of induction is shown inblack. CGV™ refers to a CRISPR Guided Vector™, which is a nucleic acidvector comprising nucleotide sequences encoding CRISPR/Cas components.

FIGS. 3A-3B. Time-kill curves for E. coli MG1655 harboring the CGV.(FIG. 3A) CRISPR induction killed 99% of the population in 60 minutes(dashed line). Growth in absence of induction is shown in black lines.CRISPR/Cas was induced at time-point 0 and monitored until 120 minutes.(FIG. 3B) Dilution series (10¹-10⁶) of drop spots (5 μl) on SM agarplates of E. coli MG1655 after 60 minutes of induction.

FIGS. 4A-4B. Specific killing of E. coli MG1655 with type I-B CRISPR-Cassystem of C. difficile in a synthetic microbial consortium. (FIG. 4A)Bacteria count of a synthetic population composed of three differentstrains. CRISPR was induced at time-point 0 and monitored for 60minutes. Growth in absence of induction is shown in black. CRISPRinduction prompted 1-log₁₀ reduction in viable cells of target strain E.coli MG1655, while leaving E. coli Top10 and L. lactis NZ9000populations intact (dark grey bars). (FIG. 4B) Dilution series (10¹-10⁶)of drop spots (5 μl) of the bacterial community mixture after 60 minutesof induction. E. coli MG1655 grows selectively on BHI+streptomycin, E.coli Top10 on ampicillin, and L. lactis NZ9000 on chloramphenicol.

FIGS. 5A-5B. Killing of E. coli MG1655 with type I-B CRISPR-Cas systemof C. difficile in a synthetic microbial consortium compared to a pureculture of E. coli MG1655. (FIG. 5A) CRISPR induction generated 4-log₁₀reductions in viable cells of target strain E. coli MG1655, both in thepure culture and in the community mixture (grey bars). Growth in absenceof induction is shown in black. (FIG. 5B) Dilution series of a pureculture of E. coli MG1655 and the bacterial community mixture onstreptomycin plates with and without inducers.

FIGS. 6A-6B. Type I CRISPR-Cas system of E. coli targeting E. coliMG1655. (FIG. 6A) Dilution series (10¹-10⁶) of drop spots (5 μl) of E.coli MG1655 harboring the CGV on SM agar plates with and withoutinducers. (FIG. 6B) CRISPR induction killed 99% of the population (greybar). Growth in absence of induction is shown in black. CGV™ refers to aCRISPR Guided Vector™, which is a nucleic acid vector comprisingnucleotide sequences encoding CRISPR/Cas components.

DETAILED DESCRIPTION

The invention relates to the production and use of Cas-encodingsequences and vectors comprising these. Aspects of the invention provideproducts, vectors, delivery vehicles, uses and methods for producingCas-encoding sequences in bacterial or archaeal cells.

An aspect of the invention provides for the control of expression of Casand optionally also Cascade proteins from single vectors, such as byregulated use of Cas modules in an operon and/or using attenuatedpromoters.

Concepts:

An aspect of the invention provides nucleic acid vectors that are usefulfor introducing into target host cells of any eukaryotic or prokaryoticspecies (eg, ex vivo or in vitro) for expressing Type I Cas andoptionally other components of a Type I CRISPR/Cas system. Usefully, thevector may in some examples therefore provide a single-vector means forintroducing a complete exogenous Type I CRISPR/Cas system into a targetcell for modification (eg, cutting by Cas3) of DNA in the target cell.In an example, a chromosomal target sequence (ie, protospacer that iscognate with the Cas3) is modified. In another example, an episomal DNAsequence is modified, for example a plasmid sequence or a DNA that hasbeen introduced into the cell. The latter may be useful in arecombineering method of the invention wherein exogenous DNA in thetarget cell is cut by the Cas3 and optionally this produces one or morerecombinogenic ends for recombination of the cut DNA with a further DNAof interest, thereby producing a recombination product in the cell. Forexample, in such a recombineering method, the target cell is arecombinongenic E coli cell, eg, comprising a red/ET system. In anotherexample, the target cell is an undesired cell (eg, a cell of a speciesor strain that is pathogenic to humans or animals, such as a bacterialdisease-causing species or strain) and the cutting by Cas3 kills thecell. This may be useful for treating or preventing an infection in ahuman or animal harbouring target cells. The provision of single-vectormeans that express minimally a Cas endonuclease (eg, Cas3), cognateaccessory proteins (eg, Cascade proteins) and at least one CRISPR array(or nucleotide sequence encoding a guide RNA (eg, a single guide RNA)),wherein the Cas, accessory proteins and array (or nucleotide sequence)comprise a functional CRISPR/Cas system is more convenient and theinventors believe more efficient for introducing into a target cell andeffecting CRISPR/Cas modification of a target sequence therein than theuse of 2 or 3 or more separate vectors (eg, a vector encoding the Casnuclease and a different vector encoding the accessory proteins, andpossibly a further vector comprising the array (or gRNA-encodingnucleotide sequence) which all need to transform the target cell for thesystem to function). This may provide one or more benefits, therefore,such as simplifying delivery (and thus the design of delivery vehicles),simplifying construction of the vector and vehicle and/or providing forbetter cutting or killing efficiencies. Conveniently, an example of theinvention therefore uses an operon for the coordinated expression in thetarget cells of the Cas and accessory proteins (and optionally also thearray or gRNA-encoding sequence(s)). Whilst not wishing to be bound byany particular theory, the introduction of a single vector (eg, using anoperon) as per the invention may advantageously coordinate theexpression of the Cas and accessory proteins (and optionally productionof cRNAs or gRNAs) so that these are available to operate togetherwithout undue delay in the target cell. This may be important to tip thebalance between, on the one hand the target cell using its endogenousanti-restriction, endogenous Cas or other endogenous mechanisms thatseek out and degrade invading phage and DNA, and on the other handefficient cell killing or deactivation of such mechanisms by theinvading CRISPR components of the vector of the invention. In such anarms race, concerted and early operation of the CRISPR components in thecell are likely to be important to gain the upper hand and effect cellkilling. The invention provides means to assist this.

By way of example, the invention thus provides the following Concepts:

-   -   1. A nucleic acid vector for introduction into a host cell, the        vector comprising a first nucleotide sequence encoding a Type I        Cas3 and a second nucleotide sequence encoding one or more        Cascade proteins, wherein the first and second sequences are        under the control of one or more promoters comprised by the        vector for expression of the proteins in the cell.    -   2. The vector of concept 1, wherein the vector comprises an        operon for expression in the cell of the Cas3 and Cascade        proteins from a Cas module, the module comprising the nucleotide        sequences encoding the Cas3 and Cascade proteins, and the operon        comprising the Cas module under the control of a promoter for        controlling the expression of both the Cas3 and Cascade        proteins.    -   3. The vector of concept 2, wherein        -   (a) the first sequence is between the promoter and the            second sequence in the operon;        -   (b) the operon comprises no Cas-encoding nucleotide            sequences between the promoter and the first nucleotide            sequence; and/or        -   (c) the operon comprises (in 5′ to 3′ direction) the            promoter, the first sequence and the second sequence.    -   4. The vector of any preceding concept, wherein each promoter is        a constitutive promoter.    -   5. The vector of any one of concepts 1 to 3, wherein the        promoter is repressible (optionally repressible by a        tetracycline repressor or lac repressor).    -   6. The vector of any one of concepts 1 to 3, wherein the        promoter is inducible.    -   7. The vector of any preceding concept, wherein the first        sequence is under the control of a medium strength promoter.    -   8. The vector of any preceding concept, wherein the first        sequence is under the control of a promoter that has an Anderson        Score (AS) of 0.5>AS>0.1.    -   9. The vector of any preceding concept, wherein the first        sequence (and optionally the second sequence) is under the        control of a promoter and translation initiation site (TIS)        combination that is capable of producing expression of green        fluorescent protein (GFP) from a first expression operating unit        (EOU) in E. coli strain BW25113 cells with a fluorescence of        from 0.5 to 4 times the fluorescence produced in E. coli strain        BW25113 cells using a second EOU comprising a P10 promoter (SEQ        ID NO: 1) combined with a BCD14 TIS (SEQ ID NO: 2), wherein the        EOUs differ only in their promoter and TIS combinations, wherein        each EOU comprises (in 5′ to 3′ direction) an upstream        initiator, the respective promoter, the respective TIS, a        nucleotide sequence encoding GFP, a 3′ UTR, a transcription        terminator and a downstream insulator.    -   10. The vector of concept 9, wherein fluorescence using the        first EOU is 0.5 to 2 times the fluorescence using the second        EOU.    -   11. The vector of any preceding concept, wherein the vector        comprises an origin of replication that is operable in the host        cell.    -   12. The vector of any preceding concept, wherein the vector        comprises an origin of replication that is operable in a        bacterial cell of a vector production strain, wherein the Cas3        is not operable in the production strain cell to target and cut        a chromosomal sequence thereof.    -   13. The vector of concept 12, wherein the first sequence is        under the control of a promoter that is capable of controlling        expression of the Cas3 at a level that is not toxic to the        production strain cell.    -   14. The vector of any preceding concept, wherein the vector is a        high copy number vector.    -   15. The vector of any preceding concept, wherein the first        nucleotide sequence or operon is comprised by a mobile genetic        element.    -   16. The vector of any preceding concept, wherein the vector is        devoid of a Cas adaption module.    -   17. The vector of any preceding concept, wherein the vector is        devoid of nucleotide sequence encoding one, more or all of a        Cas1, Cas2, Cas4, Cas6, Cas7 and Cas 8.    -   18. The vector of any preceding concept, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas11, Cas7 and Cas8a1.    -   19. The vector of concept 18, wherein the vector comprises        nucleotide sequence encoding Cas3′ and/or Cas3″.    -   20. The vector or concept 19, wherein the nucleotide sequences        encoding the Cas3′ and/or Cas3″ are between the promoter and the        sequence(s) recited in concept 18.    -   21. The vector of any one of concepts 18 to 20, wherein the host        cell comprises a Type IA CRISPR array that is cognate with the        Cas3.    -   22. The vector of any one of concepts 18 to 20, wherein the host        cell comprises an endogenous Type IB, C, U, D, E or F CRISPR/Cas        system.    -   23. The vector of any one of concepts 1 to 17, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8b1, Cas7 and Cas5.    -   24. The vector of concept 23, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in concept 23.    -   25. The vector of concept 23 or 24, wherein the host cell        comprises a Type IB CRISPR array that is cognate with the Cas3.    -   26. The vector of concept 23 or 24, wherein the host cell        comprises an endogenous Type IA, C, U, D, E or F CRISPR/Cas        system.    -   27. The vector of any one of concepts 1 to 17, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas5, Cas8c and Cas7.    -   28. The vector of concept 27, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in concept 27.    -   29. The vector of concept 27 or 28, wherein the host cell        comprises a Type IC CRISPR array that is cognate with the Cas3.    -   30. The vector of concept 27 or 28, wherein the host cell        comprises an endogenous Type IA, B, U, D, E or F CRISPR/Cas        system.    -   31. The vector of any one of concepts 1 to 17, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8U2, Cas7, Cas5 and        Cas6.    -   32. The vector of concept 31, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in concept 31.    -   33. The vector of concept 31 or 32, wherein the host cell        comprises a Type IU CRISPR array that is cognate with the Cas3.    -   34. The vector of concept 31 or 32, wherein the host cell        comprises an endogenous Type IA, B, C, D, E or F CRISPR/Cas        system.    -   35. The vector of any one of concepts 1 to 17, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas10d, Cas7 and Cas5.    -   36. The vector of concept 35, wherein the vector comprises a        nucleotide sequence encoding Cas3′ and/or Cas3″.    -   37. The vector of concept 36, wherein the nucleotide sequences        encoding the Cas3′ and/or Cas3″ are between the promoter and the        sequence(s) recited in concept 35.    -   38. The vector of any one of concepts 35 to 37, wherein the host        cell comprises a Type ID CRISPR array that is cognate with the        Cas3.    -   39. The vector of any one of concepts 35 to 37, wherein the host        cell comprises an endogenous Type IA, B, C, U, E or F CRISPR/Cas        system.    -   40. The vector of any one of concepts 1 to 17, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8e, Cas11, Cas7, Cas5        and Cas6.    -   41. The vector of concept 40, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in concept 40.    -   42. The vector of concept 40 or 41, wherein the host cell        comprises a Type IE CRISPR array that is cognate with the Cas3.    -   43. The vector of concept 40 or 41, wherein the host cell        comprises an endogenous Type IA, B, C, D, U or F CRISPR/Cas        system.    -   44. The vector of any one of concepts 1 to 17, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8f, Cas5, Cas7 and        Cas6f.    -   45. The vector of concept 44, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in concept 44, wherein the vector is devoid        of nucleotide sequence encoding further Cas between the promoter        and the sequence encoding the Cas3.    -   46. The vector of concept 44 or 45, wherein the host cell        comprises a Type IF CRISPR array that is cognate with the Cas3.    -   47. The vector of concept 44 or 45, wherein the host cell        comprises an endogenous Type IA, B, C, D, U or E CRISPR/Cas        system.    -   48. The vector of any one of concepts 1 to 17, wherein the Cas        and Cascade are        -   (a) Type IA Cas and Cascade proteins;        -   (b) Type IB Cas and Cascade proteins;        -   (c) Type IC Cas and Cascade proteins;        -   (d) Type ID Cas and Cascade proteins;        -   (e) Type IE Cas and Cascade proteins;        -   (f) Type IF Cas and Cascade proteins; or        -   (g) Type IU Cas and Cascade proteins.    -   49. The vector of any preceding concept, wherein the Cas and        Cascade are E coli (optionally Type IE or IF) Cas and Cascade        proteins.    -   50. The vector of concept 49, wherein the E coli is        ESBL-producing E. coli or E. coli ST131-O25b:H4.    -   51. The vector of any preceding concept, wherein the Cas and        Cascade are        -   (a) Clostridium (eg, C difficile) Cas and Cascade proteins,            optionally C difficile resistant to one or more antibiotics            selected from aminoglycosides, lincomycin, tetracyclines,            erythromycin, clindamycin, penicillins, cephalosporins and            fluoroquinolones;        -   (b) Pseudomonas aeruginosa Cas and Cascade proteins,            optionally P aeruginosa resistant to one or more antibiotics            selected from carbapenems, aminoglycosides, cefepime,            ceftazidime, fluoroquinolones, piperacillin and tazobactam;            or        -   (c) Klebsiella pneumoniae (eg, carbapenem-resistant            Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase            (ESBL)-producing K pneumoniae) Cas and Cascade proteins.    -   52. The vector of any preceding concept, wherein the Cas and        Cascade are E coli, C difficile, P aeruginosa, K pneumoniae, P        furiosus or B halodurans Cas and Cascade proteins.    -   53. The vector of any preceding concept, wherein the Cas3 is a        Cas3 of a CRISPR/Cas locus of a first bacterial or archaeal        species, wherein the distance between the Cas3-encoding sequence        of the locus and its cognate promoter is further than the        distance between the Cas3-encoding sequence and the respective        promoter comprised by the vector.    -   54. The vector of any preceding concept, wherein the distance        between the promoter and the Cas3-encoding sequence and/or        Cascade protein-encoding sequence(s) is shorter than in a        corresponding wild-type Type I locus.    -   55. The vector of any preceding concept, wherein the vector        comprises (i) a CRISPR array for producing crRNAs in the host        cell and/or (ii) one or more nucleotide sequences encoding one        or more guide RNAs (gRNAs or single gRNAs), wherein the crRNAs        or gRNAs are cognate to the Cas3 (and optionally cognate to the        Cascade proteins).    -   56. The vector of concept 55 when dependent from concept 2,        wherein the array or gRNA-encoding sequence(s) are comprised by        the operon and under the control of the promoter.    -   57. The vector of concept 56, wherein the array or gRNA-encoding        sequence(s) are under the control of a promoter that is        different from the promoter that controls the expression of the        Cas3.    -   58. The vector of concept 56 or 57, wherein one or more of the        crRNAs or gRNAs comprises a spacer sequence that is capable of        hybridising to a target nucleotide sequence of the host cell,        wherein the target sequence is adjacent a PAM, the PAM being        cognate to the Cas3.    -   59. The vector of concept 58, wherein the target sequence is a        chromosomal sequence of the host cell.    -   60. The vector of concept 58 or 59, wherein the Cas3 is operable        to cut the target sequence.    -   61. The vector of any preceding concept, wherein the vector is a        plasmid or phagemid.    -   62. A delivery vehicle comprising the vector of any preceding        concept, wherein the delivery vehicle is capable of delivering        the vector into the host cell.    -   63. The vehicle of concept 62, wherein the delivery vehicle is a        phage, non-replicative transduction particle, nanoparticle        carrier, bacterium or liposome.    -   64. The vector or vehicle of any preceding concept, wherein the        host cell is a bacterial or archaeal cell, optionally, the host        cell is a C difficile, P aeruginosa, K pneumoniae (eg,        carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum        Beta-Lactamase (ESBL)-producing K pneumoniae), E coli (eg,        ESBL-producing E. coli, or E. coli ST131-O25b:H4), H pylori, S        pneumoniae or S aureus cell.    -   65. The vector or vehicle of any preceding concept for        administration to a human or animal subject for treating or        reducing the risk of a disease or condition in the subject.    -   66. The vector or vehicle of concept 65, wherein the disease or        condition is an infection of the subject with host cells (eg,        bacterial cells), or wherein the disease or condition is        mediated by host cells (eg, bacterial cells).    -   67. A pharmaceutical composition comprising the vector or        vehicle of any preceding concept and a pharmaceutically        acceptable diluent, excipient or carrier.    -   68. A method of treating or reducing the risk of a disease or        condition in a human or animal subject, the method comprising        administering the vector, vehicle or composition of any        preceding concept to the subject, and introducing the vector        into target host bacterial or archaeal cells in the subject (eg,        in a gut microbiota, lung, eye or blood of the subject), wherein        the Cas cuts (or otherwise modifies) one or more target        sequences in the target cells and the cells are killed or growth        or proliferation of the cells is reduced.    -   69. The method of concept 68, wherein the target cells are cells        of a disease pathogen species.    -   70. The method of concept 68 or 69, wherein the target cells are        C difficile, P aeruginosa, K pneumoniae (eg,        carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum        Beta-Lactamase (ESBL)-producing K pneumoniae), E coli (eg,        ESBL-producing E. coli, or E. coli ST131-O25b:H4), H pylori, S        pneumoniae or S aureus cells.

EMBODIMENTS

An aspect of the invention provides improved ways of amplifying DNAconstructs in bacterial and archaeal production strain cells. Forexample, the DNA may be a high copy number plasmid or phagemidcomprising a constitutive promoter for controlling the expression of oneor more Cas proteins when the DNA has been introduced into a target hostbacterial or host cell. It is desirable, according to an aspect of theinvention, to consider attenuating the promoter activity duringamplification of the DNA in the production strain. This is useful, sincethe inventors have found that Cas expression in production strains maybe toxic to production strain cells, thereby reducing the yield ofamplified DNA. Toxicity may be due, for example, to off-target cuttingof the production strain chromosomal DNA when the Cas is a nuclease(such as Cas9 or Cas3) and/or due to relatively high levels ofexpression of the Cas in the cells. Additionally or alternatively,undesirably the Cas expression or activity may impose a selectivepressure that favours mutation and propagation of mutated DNA constructs(such as mutation in one more or all of a CRISPR/Cas operon,Cas-encoding gene, Cascade-encoding gene, CRISPR array and gRNa-encodingsequence of the DNA construct) in production cells, thereby reducing theyield of desired amplified constructs and imposing an undesired step ofseparating desired from mutated DNA constructs for further formulationinto useful compositions. Such compositions may be pharmaceuticalcompositions, herbicides, pesticides, environmental remediationcompositions etc. In one example, the promoter attenuation in productionstrains is achieved by using a medium strength (not high or low)promoter to control the Cas-encoding nucleotide sequence of the DNAconstructs. A medium level of Cas expression may be tolerable in theproduction strains, and yet once the DNA is subsequently introduced intotarget host cells the Cas is expressed at sufficiently high levels toproduce desired activity to modify (eg, cut) target sequences in targetcells. In an alternative, the invention uses a repressible promoter,wherein the promoter is repressed in production strain, but notrepressed in target host cells. For example, aspects of the inventionuse a tetracycline repressor (tetR) expressed in production strain cellsthat represses the promoter.

Thus, the yield can be enhanced by one or more of

-   -   (a) reducing toxicity of the Cas in the production strain;    -   (b) reducing mutation of the DNA (optionally the Cas-encoding        sequence) in the production strain;    -   (c) promoting production cell viability during the amplification        of the DNA; and    -   (d) reducing the occurrence of Cas cutting of DNA (optionally        cutting of production host cell chromosomal DNA or said DNA        construct).

To this end, the invention provides Embodiments as follows:

-   -   1. A method of amplifying copies of a DNA encoding a functional        Cas protein (optionally a Cas nuclease) in a bacterial or        archaeal production strain of cells, the method comprising        -   (a) Providing production strain cells, each cell comprising            a copy of said DNA, wherein each DNA comprises a nucleotide            sequence encoding said Cas, wherein the nucleotide sequence            is under the control of a promoter for controlling the            expression of the Cas in the production strain cell, the DNA            comprising an origin of replication that is operable in the            cell for replication of the DNA;        -   (b) Culturing the cells to allow replication of the DNA,            whereby the DNA is amplified; and        -   (c) Optionally isolating copies of the DNA,        -   wherein the promoter is an attenuated constitutive promoter.    -   In an example, promoter is a medium strength promoter. In        another example, the promoter is repressed in the production        strain cell. Hence, the promoter is an attenuated promoter in        these examples.    -   2. Use of an attenuated promoter in a DNA construct comprising a        nucleotide sequence encoding a functional Cas protein        (optionally a Cas nuclease) that is under the control of the        promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for enhancing        the yield of amplified DNA produced by the production host        cells.    -   3. The use of paragraph 2, wherein the use is for enhancing said        yield by        -   (a) reducing toxicity of the Cas in the production strain;        -   (b) reducing mutation of the DNA (optionally the            Cas-encoding sequence) in the production strain;        -   (c) promoting production cell viability during the            amplification of the DNA; and/or        -   (d) reducing the occurrence of Cas cutting of DNA            (optionally cutting of production host cell chromosomal DNA            or said DNA construct).    -   4. Use of an attenuated promoter in a DNA construct comprising a        nucleotide sequence encoding a functional Cas protein        (optionally a Cas nuclease) that is under the control of the        promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for reducing        toxicity of the Cas in the production strain.    -   5. Use of an attenuated promoter in a DNA construct comprising a        nucleotide sequence encoding a functional Cas protein        (optionally a Cas nuclease) that is under the control of the        promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for reducing        mutation of the DNA (optionally the Cas-encoding sequence) in        the production strain.    -   6. Use of an attenuated promoter in a DNA construct comprising a        nucleotide sequence encoding a functional Cas protein        (optionally a Cas nuclease) that is under the control of the        promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for promoting        production cell viability during the amplification of the DNA.    -   7. Use of an attenuated promoter in a DNA construct comprising a        nucleotide sequence encoding a functional Cas protein        (optionally a Cas nuclease) that is under the control of the        promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for reducing        the occurrence of Cas cutting of DNA.    -   8. A method for enhancing the yield of amplified copies of a DNA        construct in a population of bacterial or archaeal production        strain cells, wherein the construct comprises a nucleotide        sequence encoding a functional Cas protein (optionally a Cas        nuclease) that is under the control of a promoter, the method        comprising culturing the cells to allow replication of the DNA        thereby amplifying the DNA in the cells, wherein the promoter is        an attenuated promoter.    -   9. A method for reducing toxicity of a functional Cas protein        (optionally a Cas nuclease) in a population of bacterial or        archaeal production strain cells in a process of amplifying        copies of a DNA construct, wherein the construct comprises a        nucleotide sequence encoding the Cas and the sequence is under        the control of a promoter, the method comprising culturing the        cells to allow replication of the DNA thereby amplifying the DNA        in the cells, wherein the promoter is an attenuated promoter.    -   10. A method for reducing mutation of a DNA construct encoding a        functional Cas protein (optionally a Cas nuclease) in a        population of bacterial or archaeal production strain cells in a        process of amplifying copies of the construct, wherein the        construct comprises a nucleotide sequence encoding the Cas and        the sequence is under the control of a promoter, the method        comprising culturing the cells to allow replication of the DNA        thereby amplifying the DNA in the cells, wherein the promoter is        an attenuated promoter.    -   11. A method for promoting production cell viability of a        population of bacterial or archaeal production strain cells in a        process of amplifying copies of a DNA construct comprised by the        cells, wherein the construct comprises a nucleotide sequence        encoding a functional Cas protein (optionally a Cas nuclease)        and the sequence is under the control of a promoter, the method        comprising culturing the cells to allow replication of the DNA        thereby amplifying the DNA in the cells, wherein the promoter is        an attenuated promoter.    -   12. A method for reducing the occurrence of Cas nuclease cutting        of a DNA construct in a population of bacterial or archaeal        production strain cells in a process of amplifying copies of the        construct, wherein the construct comprises a nucleotide sequence        encoding the Cas and the sequence is under the control of a        promoter, the method comprising culturing the cells to allow        replication of the DNA thereby amplifying the DNA in the cells,        wherein the promoter is an attenuated promoter.    -   13. The use of paragraph 5 or 7, or the method of paragraph 10        or 12, wherein the mutation or cutting is mutation or cutting of        host cell chromosomal DNA or the construct DNA.    -   14. The method or use of any one of paragraphs 2 to 13, wherein        the promoter is a constitutive promoter.    -   15. The method or use of any preceding paragraph, wherein the        promoter is repressed in the production strain cells (optionally        repressed by a tetracycline repressor or a lac repressor).    -   16. The method or use of paragraph 15, wherein the promoter is        P_(tetO-1), P_(LlacO-1) or a repressible homologue thereof.    -   Other examples of suitable repressible promoters are Ptac        (repressed by lacI) and the Leftward promoter (pL) of phage        lambda (which repressed by the λcI repressor). In an example,        the promoter comprises a repressible operator (eg, tetO or lacO)        fused to a promoter sequence. The corresponding repressor is        encoded by a nucleic acid in the production strain (eg, a        chromosomally-integrated sequence or a sequence comprised by an        episome) and the repressor is expressed during the DNA or vector        amplification method of the invention, whereby the promoter        controlling Cas expression is repressed. In delivery vehicles        that are subsequently produced from isolated amplified        DNA/vector, the vehicle is devoid of an expressible nucleotide        sequence encoding the repressor, whereby the promoter is        functional when the DNA/vector is introduced into a target host        cell. For example, in the absence of the repressor the promoter        is constitutively ON for expression of the Cas. The system is        therefore primed to work once the DNA/vector is introduced into        the host cells, and this effect can be enhanced further by using        a high copy number DNA/vector comprising an origin of        replication that is operable in the host cell. A high copy        number vector or DNA is also desirable in the production strain        cells for enhancing yield of the DNA/vector, and by use of an        attenuated promoter as described herein (eg, medium strength        promoter and/or repressed promoter in the production strain        cells) one can minimise Cas toxicity whilst culturing to        maximise amplification and thus yield of the DNA/vector.    -   17. The method or use of any preceding paragraph, wherein the        promoter is a medium strength promoter.    -   18. The method or use of any preceding paragraph, wherein the        promoter has an Anderson Score (AS) of 0.5>AS>0.1.    -   19. The method or use of any preceding paragraph, wherein the        nucleotide sequence encoding said Cas is under the control of a        promoter and translation initiation site (TIS) combination that        is capable of producing expression of green fluorescent protein        (GFP) from a first expression operating unit (EOU) in E. coli        strain BW25113 cells with a fluorescence of from 0.5 to 4 times        the fluorescence produced in E. coli strain BW25113 cells using        a second EOU comprising a P10 promoter (SEQ ID NO: 1) combined        with a BCD14 TIS (SEQ ID NO: 2), wherein the EOUs differ only in        their promoter and TIS combinations, wherein each EOU comprises        (in 5′ to 3′ direction) an upstream initiator, the respective        promoter, the respective TIS, a nucleotide sequence encoding        GFP, a 3′ UTR, a transcription terminator and a downstream        insulator.    -   20. The method or use of paragraph 19, wherein fluorescence        using the first EOU is 0.5 to 2 times the fluorescence using the        second EOU.    -   21. The method or use of any preceding paragraph, wherein the        nuclease is Cas3 and optionally the DNA or cell encodes cognate        Cascade proteins.    -   22. The method or use of any one of paragraphs 1 to 20, wherein        the Cas is a Cas9.    -   23. The method or use of any preceding paragraph, wherein the        production strain cells comprise a helper phage genome that is        inducible to produce phage coat proteins in the cells, wherein        the method further comprises inducing production of the phage        proteins and causing packaging of the amplified DNA into phage        particles or non-self-replicative transduction particles, and        further isolating the phage or transduction particles and        optionally formulating the particles into a pharmaceutical        composition for administration to a human or animal subject for        treating or reducing the risk of a disease or condition in the        subject.    -   24. The method or use of paragraph 23, wherein the particles are        capable of infecting target host cells in the subject and        transducing the cells with the DNA, wherein the Cas and crRNAs        (or guide RNAs, gRNAs) encoded by the DNA are expressed in the        cells, the crRNAs or (gRNAs) being operable to guide the Cas to        a target nucleotide sequence (optionally a chromosomal sequence)        comprised by the cells, wherein the Cas cuts the target        sequences in the cells, thereby killing host cells and treating        or reducing the risk of the disease or condition.    -   25. The method or use of paragraph 24, wherein the host cells        are bacterial or archaeal cells, optionally, the host cells are        C difficile, P aeruginosa, K pneumoniae (eg,        carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum        Beta-Lactamase (ESBL)-producing K pneumoniae), E coli (eg,        ESBL-producing E. coli, or E. coli ST131-O25b:H4), H pylori, S        pneumoniae or S aureus cells.    -   26. The method or use of any preceding paragraph, wherein each        DNA is comprised by a high copy number plasmid or phagemid.    -   27. The method or use of any preceding paragraph, wherein the        DNA construct comprises one or more nucleotide sequences for        producing crRNAs or gRNAs that are operable for Cas nuclease        targeting in target host cells.

Paragraphs & Generally Applicable Features

The invention provides the following Paragraphs, which are supported bythe Examples below. Any features of the Concepts are combinable with anyfeatures of the Embodiments. Any features of the Concepts are combinablewith any features of the Embodiments. Any features of the Paragraphs arecombinable with any features of the Embodiments.

Any cell herein (eg, a production strain cell or target host cell) maybe a bacterial cell, archaeal cell, algal cell, fungal cell, protozoancell, invertebrate cell, vertebrate cell, fish cell, bird cell, mammalcell, companion animal cell, dog cell, cat cell, horse cell, mouse cell,rat cell, rabbit cell, eukaryotic cell, prokaryotic cell, human cell,animal cell, rodent cell, insect cell or plant cell. Preferably, thecell is a bacterial cell. Alternatively, the cell is a human cell.Optionally, the production strain cell(s) and target host cell(s) are ofthe same phylum, order, family, genus, species or strain.

-   -   1. A nucleic acid vector for introduction into a host cell, the        vector comprising a first nucleotide sequence encoding a Type I        Cas3, wherein the sequence is under the control of a promoter        comprised by the vector for expression of the Cas3 in the cell.    -   In an example, the vector is a DNA vector, eg, ssDNA vector or        dsDNA vector.    -   2. The vector of paragraph 1, wherein the vector comprises a        second nucleotide sequence encoding one or more Cascade        proteins, wherein the first and second sequences are under the        control of one or more promoters comprised by the vector for        expression of the proteins in the cell.    -   3. The vector of paragraph 2, wherein the Cascade protein(s) are        cognate with the Cas3.    -   In an example, the Cas3 is cognate with Cascade proteins encoded        by the host cell and/or encoded by a second operon. Optionally,        the second operon is comprised by the vector. Optionally, the        second operon is comprised by a second vector that is capable of        introducing the second operon into the host cell, whereby the        Cas3 and Cascade proteins are expressed from the operons in the        host cell and are operable with crRNA or gRNA to target the Cas        to a host cell target sequence, wherein the Cas3 is capable of        modifying the target sequence.    -   4. The vector of paragraph 2 or 3, wherein the vector comprises        an operon for expression in the cell of the Cas3 and Cascade        proteins from a Cas module, the module comprising the nucleotide        sequences encoding the Cas3 and Cascade proteins, and the operon        comprising the Cas module under the control of a promoter for        controlling the expression of both the Cas3 and Cascade        proteins.    -   The term “operon” is known to the skilled person such as        relating to a functioning unit of DNA containing at least        expressible 2 nucleotide sequences respectively encoding for an        expression product (eg, a respective translatable mRNA), wherein        the sequences are under common promoter control.    -   5. The vector of paragraph 4, wherein the first sequence is        between the promoter and the second sequence in the operon.    -   6. The vector of paragraph 4 or 5, wherein the operon comprises        no Cas-encoding nucleotide sequences between the promoter and        the first nucleotide sequence.    -   Optionally, the Cas3 is a Cas3 encoded by a CRISPR/Cas locus of        a first bacterial or archaeal species, wherein in the locus the        Cas3-encoding sequence is 3′ of Cascade protein-encoding        sequences (ie, the latter are between the Cas3 and the 5′-most        promoter of the locus).    -   Optionally, the Cas3 is a ygcB protein (eg, wherein the        production strain cell and/or host target cell is an E coli).    -   Optionally, the Cascade proteins comprise or consist of    -   cas5 (casD, csy2)    -   cas6 (cas6f, cse3, casE)    -   cas7 (csc2, csy3, cse4, casC)    -   cas8 (casA, cas8a1, cas8b1, cas8c, cas10d, cas8e, cse1, cas8f,        csy1).    -   Optionally herein the promoter and the Cas3-encoding sequence        are spaced no more than 150, 100, 50, 40, 30, 20 or 10 bp apart,        eg, from 30-45, or 30-40, or 39 or around 39 bp apart.    -   Optionally herein a ribosome binding site and the Cas3-encoding        sequence are spaced no more than 20, 15, 14, 13, 12, 11, 10, 9,        8, 7, 6, 4 or 3 bp apart, eg, from 10-5, 6 or around 6 bp apart.    -   7. The vector of any one of paragraphs 4 to 6, wherein the        operon comprises (in 5′ to 3′ direction) the promoter, the first        sequence and the second sequence.    -   8. The vector of any preceding paragraph, wherein each promoter        is a constitutive promoter.    -   9. The vector of any one of paragraphs 1 to 7, wherein the        promoter is repressible (optionally repressible by a        tetracycline repressor or lac repressor).    -   10. The vector of any one of paragraphs 1 to 7, wherein the        promoter is inducible.    -   11. The vector of any preceding paragraph, wherein the first        sequence is under the control of a weak promoter.    -   12. The vector of any one of paragraphs 1 to 7, wherein the        first sequence is under the control of a medium strength        promoter.    -   13. The vector of any one of paragraphs 1 to 7, wherein the        first sequence is under the control of a strong promoter.    -   In an example, the promoter is in combination with a        Shine-Dalgarno sequence comprising the sequence        5′-aaagaggagaaa-3′ (SEQ ID NO: 5) or a ribosome binding site        homologue thereof.    -   14. The vector of any one of paragraphs 1 to 7, wherein the        first sequence is under the control of a promoter that has an        Anderson Score (AS) of AS≥0.5.    -   See Table 2 for more information on Anderson Scores in relation        to promoters.    -   15. The vector of any one of paragraphs 1 to 7, wherein the        first sequence is under the control of a promoter that has an        Anderson Score (AS) of 0.5>AS>0.1.    -   16. The vector of any one of paragraphs 1 to 7, wherein the        first sequence is under the control of a promoter that has an        Anderson Score (AS) of ≤0.1.    -   17. The vector of any one of paragraphs 1 to 7, wherein the        first sequence (and optionally the second sequence) is under the        control of a promoter and translation initiation site (TIS)        combination that is capable of producing expression of green        fluorescent protein (GFP) from a first expression operating unit        (EOU) in E. coli strain BW25113 cells with a fluorescence of        from 0.5 to 4 times the fluorescence produced in E. coli strain        BW25113 cells using a second EOU comprising a P10 promoter (SEQ        ID NO: 1) combined with a BCD14 TIS (SEQ ID NO: 2), wherein the        EOUs differ only in their promoter and TIS combinations, wherein        each EOU comprises (in 5′ to 3′ direction) an upstream        initiator, the respective promoter, the respective TIS, a        nucleotide sequence encoding GFP, a 3′ UTR, a transcription        terminator and a downstream insulator.    -   18. The vector of paragraph 17, wherein fluorescence using the        first EOU is 0.5 to 2 times the fluorescence using the second        EOU.    -   For example, fluorescence using the first EOU is 0.5 to X times        the fluorescence using the second EOU, wherein X is from 3.0 to        1.0, eg, 3, 2.5, 2, 1.5 or 1, wherein fluorescence is determined        using excitation at 481 nm and emission at 507 nm. Optionally, E        coli cultures at OD600 of 0.3-0.5 in the exponential growth        phase are used.    -   For example, the upstream insulator, the nucleotide sequence        encoding GFP, 3′ UTR, transcription terminator and downstream        insulator of each EOU are as disclosed in Mutalik et al (2013).        For example, the upstream insulator, the nucleotide sequence        encoding GFP, 3′ UTR, transcription terminator and downstream        insulator of each EOU are corresponding sequences of SEQ ID        NO: 4. For example, the E coli is E. coli BW25113 is grown in        MOPS EZ Rich Medium (Teknova) supplemented with 50 μg/ml        kanamycin (kan) at 37° C., shaken at 900 r.p.m. For example,        each EOUs is comprised by a medium copy plasmid, eg, a plasmid        derived from pFAB217 comprising a p15A replication origin and a        kan resistance gene.    -   19. The vector of any preceding paragraph, wherein the vector        comprises an origin of replication that is operable in the host        cell.    -   20. The vector of any preceding paragraph, wherein the vector        comprises an origin of replication that is operable in a        bacterial cell of a vector production strain, wherein the Cas3        is not operable in the production strain cell to target and cut        a chromosomal sequence thereof.    -   An example of a production strain cell is an E coli cell. A        production strain cell is a cell that is used to amplify DNA        encoding Cas (and optionally other components of a CRISPR/Cas        system). Usefully, the strain may package the amplified DNA into        transduction particles that are may be isolated to produce a        composition that can be contacted with a population of target        host cells (eg, bacterial, archaeal, prokaryotic, eukaryotic,        human, animal, mammal, rodent, mouse, rat, rabbit, Xenopus,        fish, bird, amphibian, insect, plant, amoeba or algae cells)        wherein the DNA is introduced into the cells for expression of        the Cas (and optional other CRISPR/Cas system components),        wherein the Cas is guided to a protospacer target sequence in        the host cells and modifies (eg, cuts) the sequence. In another        example, the amplified DNA isolated from a population of        production strain cells and is combined with a delivery vehicle        (eg, a carrier bacterium, nanoparticle or liposome), wherein the        delivery vehicle can be contacted with a population of target        host cells (eg, bacterial, archaeal, prokaryotic, eukaryotic,        human, animal, mammal, rodent, mouse, rat, rabbit, Xenopus,        fish, bird, amphibian, insect, plant, amoeba or algae cells)        wherein the DNA is introduced into the cells for expression of        the Cas (and optional other CRISPR/Cas system components),        wherein the Cas is guided to a protospacer target sequence in        the host cells and modifies (eg, cuts) the sequence.    -   21. The vector of paragraph 20, wherein the first sequence is        under the control of a promoter that is capable of controlling        expression of the Cas3 at a level that is not toxic to the        production strain cell.    -   In an example, substantially no production strain cells are        killed when the Cas3-encoding sequence is amplified therein. In        another example, no more than 40, 30, 20, 10, 5, 4, 3, 2, or 1%        of production strain cells are killed when the Cas3-encoding        sequence is amplified therein. For example this is in a 1, 2, 3,        4, 5, 6, 7, 8 9 10, 12 or 24 hour period of culturing the cells.    -   22. The vector of paragraph 20, wherein the first sequence is        under the control of a promoter that controls expression of the        Cas3 in the production strain cell such that the cell is capable        of growth and propagation sufficient to produce at least 1000        copies of the vector.    -   For example this is in a 1, 2, 3, 4, 5, 6, 7, 8 9 10, 12 or 24        hour period of culturing the cells. For example, at least 10⁴,        10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,        10¹⁶, 10¹⁷ or 10¹⁸ copies of the vector are produced per 10³,        10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴,        10¹⁵, 10¹⁶, 10¹⁷ production strain cells respectively.    -   23. The vector of any one of paragraphs 20 to 22, wherein the        cell is capable of at least 2 or 3 logs of expansion when the        vector is comprised therein.    -   For example, this is in a 1, 2, 3, 4, 5, 6, 7, 8 9 10, 12 or 24        hour period of culturing the cells.    -   24. The vector of any preceding paragraph, wherein the vector is        a high copy number vector.    -   25. The vector of any preceding paragraph, wherein the first        nucleotide sequence or operon is comprised by a mobile genetic        element.    -   Suitable mobile genetic elements, eg, transposons, are disclosed        in WO2016177682 and US20170246221, the disclosures of which are        explicitly incorporated herein for possible use in the invention        and for providing one or more features for the claims herein.    -   26. The vector of any preceding paragraph, wherein the vector is        devoid of a Cas adaption module. For example, the vector is        devoid of nucleotide sequences encoding a Cas1, Cas2 and/or        Cas4.    -   27. The vector of any preceding paragraph, wherein the vector is        devoid of nucleotide sequence encoding one, more or all of a        Cas1, Cas2, Cas4, Cas6 (optionally Cas6f), Cas7 and Cas 8        (optionally Cas8f).    -   28. The vector of any preceding paragraph, wherein the vector is        devoid of a sequence encoding a Cas6 (optionally a Cas6f).    -   29. The vector of any one of paragraphs 1 to 27, wherein the        module encodes a Cas6 (optionally a Cas6f).    -   30. The vector of any preceding paragraph, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas 11, Cas7 and Cas8a1.    -   31. The vector of paragraph 30, wherein the vector comprises        nucleotide sequence encoding Cas3′ and/or Cas3″ (optionally        wherein the nucleotide sequences encoding the Cas3′ and/or Cas3″        are between the promoter and the sequence(s) recited in        paragraph 30).    -   In one embodiment, the vector comprises nucleotide sequences (in        5′ to 3′ direction) that encode a Cas3 (eg, Cas3′ and/or Cas3″),        Cas11, Cas7 and Cas8a1. Optionally, a nucleotide sequence        encoding Cas6 is between the Cas3 sequence(s) and the Cas11        sequence. Optionally, the vector comprises a Type IA CRISPR        array or one or more nucleotide sequences encoding single guide        RNA(s) (gRNA(s)), wherein the array and each gRNA comprises        repeat sequence that is cognate with the Cas3. Thus, the array        is operable in a host cell when the vector has been introduced        into the cell for production of guide RNAs, wherein the guide        RNAs are operable with the Cas and Cascade proteins to target        and modify (eg, cut) a target nucleotide sequence in the host        cell, optionally thereby killing the host cell. Similarly, the        single guide RNAs encoded by the vector in one embodiment are        operable with the Cas and Cascade proteins to target and modify        (eg, cut) a target nucleotide sequence in the host cell,        optionally thereby killing the host cell.    -   32. The vector of paragraph 30 or 31, wherein the host cell        comprises a Type IA CRISPR array that is cognate with the Cas3.    -   33. The vector of paragraph 30 or 31, wherein the host cell        comprises an endogenous Type IB, C, U, D, E or F CRISPR/Cas        system.    -   34. The vector of any one of paragraphs 1 to 29, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8b1, Cas7 and Cas5.    -   35. The vector of paragraph 34, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in paragraph 34.    -   In one embodiment, the vector comprises nucleotide sequences (in        5′ to 3′ direction) that encode a Cas3, Cas8b1, Cas7 and Cas5.        Optionally, a nucleotide sequence encoding Cas6 is between the        Cas3 sequence(s) and the Cas8b1 sequence. Optionally, the vector        comprises a Type IB CRISPR array or one or more nucleotide        sequences encoding single guide RNA(s) (gRNA(s)), wherein the        array and each gRNA comprises repeat sequence that is cognate        with the Cas3. Thus, the array is operable in a host cell when        the vector has been introduced into the cell for production of        guide RNAs, wherein the guide RNAs are operable with the Cas and        Cascade proteins to target and modify (eg, cut) a target        nucleotide sequence in the host cell, optionally thereby killing        the host cell. Similarly, the single guide RNAs encoded by the        vector in one embodiment are operable with the Cas and Cascade        proteins to target and modify (eg, cut) a target nucleotide        sequence in the host cell, optionally thereby killing the host        cell.    -   36. The vector of paragraph 34 or 35, wherein the host cell        comprises a Type IB CRISPR array that is cognate with the Cas3.    -   37. The vector of paragraph 34 or 35, wherein the host cell        comprises an endogenous Type IA, C, U, D, E or F CRISPR/Cas        system.    -   38. The vector of any one of paragraphs 1 to 29, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas5, Cas8c and Cas7.    -   39. The vector of paragraph 38, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in paragraph 38.    -   In one embodiment, the vector comprises nucleotide sequences (in        5′ to 3′ direction) that encode a Cas3, Cas5, Cas8c and Cas7.        Optionally, a nucleotide sequence encoding Cas6 is between the        Cas3 sequence(s) and the Cas5 sequence. Optionally, the vector        comprises a Type IC CRISPR array or one or more nucleotide        sequences encoding single guide RNA(s) (gRNA(s)), wherein the        array and each gRNA comprises repeat sequence that is cognate        with the Cas3. Thus, the array is operable in a host cell when        the vector has been introduced into the cell for production of        guide RNAs, wherein the guide RNAs are operable with the Cas and        Cascade proteins to target and modify (eg, cut) a target        nucleotide sequence in the host cell, optionally thereby killing        the host cell. Similarly, the single guide RNAs encoded by the        vector in one embodiment are operable with the Cas and Cascade        proteins to target and modify (eg, cut) a target nucleotide        sequence in the host cell, optionally thereby killing the host        cell.    -   40. The vector of paragraph 38 or 39, wherein the host cell        comprises a Type IC CRISPR array that is cognate with the Cas3.    -   41. The vector of paragraph 38 or 39, wherein the host cell        comprises an endogenous Type IA, B, U, D, E or F CRISPR/Cas        system.    -   42. The vector of any one of paragraphs 1 to 29, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8U2, Cas7, Cas5 and        Cas6.    -   43. The vector of paragraph 42, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in paragraph 42.    -   In one embodiment, the vector comprises nucleotide sequences (in        5′ to 3′ direction) that encode a Cas3, Cas8U2, Cas7, Cas5 and        Cas6. Optionally, a nucleotide sequence encoding Cas6 is between        the Cas3 sequence(s) and the Cas8U2 sequence. Optionally, the        vector comprises a Type IU CRISPR array or one or more        nucleotide sequences encoding single guide RNA(s) (gRNA(s)),        wherein the array and each gRNA comprises repeat sequence that        is cognate with the Cas3. Thus, the array is operable in a host        cell when the vector has been introduced into the cell for        production of guide RNAs, wherein the guide RNAs are operable        with the Cas and Cascade proteins to target and modify (eg, cut)        a target nucleotide sequence in the host cell, optionally        thereby killing the host cell. Similarly, the single guide RNAs        encoded by the vector in one embodiment are operable with the        Cas and Cascade proteins to target and modify (eg, cut) a target        nucleotide sequence in the host cell, optionally thereby killing        the host cell.    -   44. The vector of paragraph 42 or 43, wherein the host cell        comprises a Type IU CRISPR array that is cognate with the Cas3.    -   45. The vector of paragraph 42 or 43, wherein the host cell        comprises an endogenous Type IA, B, C, D, E or F CRISPR/Cas        system.    -   46. The vector of any one of paragraphs 1 to 29, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas10d, Cas7 and Cas5.    -   47. The vector of paragraph 46, wherein the vector comprises a        nucleotide sequence encoding Cas3′ and/or Cas3″ (optionally        wherein the nucleotide sequences encoding the Cas3′ and/or Cas3″        are between the promoter and the sequence(s) recited in        paragraph 46).    -   In one embodiment, the vector comprises nucleotide sequences (in        5′ to 3′ direction) that encode a Cas3, Cas10d, Cas7 and Cas5.        Optionally, a nucleotide sequence encoding Cas6 is between the        Cas3 sequence(s) and the Cas10d sequence. Optionally, the vector        comprises a Type ID CRISPR array or one or more nucleotide        sequences encoding single guide RNA(s) (gRNA(s)), wherein the        array and each gRNA comprises repeat sequence that is cognate        with the Cas3. Thus, the array is operable in a host cell when        the vector has been introduced into the cell for production of        guide RNAs, wherein the guide RNAs are operable with the Cas and        Cascade proteins to target and modify (eg, cut) a target        nucleotide sequence in the host cell, optionally thereby killing        the host cell. Similarly, the single guide RNAs encoded by the        vector in one embodiment are operable with the Cas and Cascade        proteins to target and modify (eg, cut) a target nucleotide        sequence in the host cell, optionally thereby killing the host        cell.    -   48. The vector of paragraph 46 or 47, wherein the host cell        comprises a Type ID CRISPR array that is cognate with the Cas3.    -   49. The vector of paragraph 46 or 47, wherein the host cell        comprises an endogenous Type IA, B, C, U, E or F CRISPR/Cas        system.    -   50. The vector of any one of paragraphs 1 to 29, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8e, Cas11, Cas7, Cas5        and Cas6.    -   51. The vector of paragraph 50, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in paragraph 50.    -   In one embodiment, the vector comprises nucleotide sequences (in        5′ to 3′ direction) that encode a Cas3, Cas8e, Cas11, Cas7, Cas5        and Cas6. Optionally, a nucleotide sequence encoding Cas6 is        between the Cas3 sequence(s) and the Cas11 sequence. Optionally,        the vector comprises a Type IE CRISPR array or one or more        nucleotide sequences encoding single guide RNA(s) (gRNA(s)),        wherein the array and each gRNA comprises repeat sequence that        is cognate with the Cas3. Thus, the array is operable in a host        cell when the vector has been introduced into the cell for        production of guide RNAs, wherein the guide RNAs are operable        with the Cas and Cascade proteins to target and modify (eg, cut)        a target nucleotide sequence in the host cell, optionally        thereby killing the host cell. Similarly, the single guide RNAs        encoded by the vector in one embodiment are operable with the        Cas and Cascade proteins to target and modify (eg, cut) a target        nucleotide sequence in the host cell, optionally thereby killing        the host cell.    -   52. The vector of paragraph 50 or 51, wherein the host cell        comprises a Type IE CRISPR array that is cognate with the Cas3.    -   53. The vector of paragraph 50 or 51, wherein the host cell        comprises an endogenous Type IA, B, C, D, U or F CRISPR/Cas        system.    -   54. The vector of any one of paragraphs 1 to 29, wherein the        vector comprises (optionally in 5′ to 3′ direction) nucleotide        sequence encoding one, more or all of Cas8f, Cas5, Cas7 and        Cas6f.    -   55. The vector of paragraph 54, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in paragraph 54, wherein the vector is        devoid of nucleotide sequence encoding further Cas between the        promoter and the sequence encoding the Cas3.    -   In one embodiment, the vector comprises nucleotide sequences (in        5′ to 3′ direction) that encode a Cas3, Cas8f, Cas5, Cas7 and        Cas6f. Optionally, a nucleotide sequence encoding Cas6 is        between the Cas3 sequence(s) and the Cas8f sequence. Optionally,        the vector comprises a Type IF CRISPR array or one or more        nucleotide sequences encoding single guide RNA(s) (gRNA(s)),        wherein the array and each gRNA comprises repeat sequence that        is cognate with the Cas3. Thus, the array is operable in a host        cell when the vector has been introduced into the cell for        production of guide RNAs, wherein the guide RNAs are operable        with the Cas and Cascade proteins to target and modify (eg, cut)        a target nucleotide sequence in the host cell, optionally        thereby killing the host cell. Similarly, the single guide RNAs        encoded by the vector in one embodiment are operable with the        Cas and Cascade proteins to target and modify (eg, cut) a target        nucleotide sequence in the host cell, optionally thereby killing        the host cell.    -   56. The vector of paragraph 54 or 55, wherein the host cell        comprises a Type IF CRISPR array that is cognate with the Cas3.    -   57. The vector of paragraph 54 or 55, wherein the host cell        comprises an endogenous Type IA, B, C, D, U or E CRISPR/Cas        system.    -   58. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Type IA Cas and Cascade proteins.    -   59. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Type IB Cas and Cascade proteins.    -   60. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Type IC Cas and Cascade proteins.    -   61. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Type ID Cas and Cascade proteins.    -   62. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Type IE Cas and Cascade proteins.    -   63. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Type IF Cas and Cascade proteins.    -   64. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Type IU Cas and Cascade proteins.    -   65. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are E coli (optionally Type IE or IF) Cas and        Cascade proteins, optionally wherein the E coli is        ESBL-producing E. coli or E. coli ST131-O25b:H4.    -   66. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Clostridium (eg, C difficile) Cas and Cascade        proteins, optionally C difficile resistant to one or more        antibiotics selected from aminoglycosides, lincomycin,        tetracyclines, erythromycin, clindamycin, penicillins,        cephalosporins and fluoroquinolones.    -   67. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Pseudomonas aeruginosa Cas and Cascade proteins,        optionally P aeruginosa resistant to one or more antibiotics        selected from carbapenems, aminoglycosides, cefepime,        ceftazidime, fluoroquinolones, piperacillin and tazobactam.    -   68. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are Klebsiella pneumoniae (eg, carbapenem-resistant        Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase        (ESBL)-producing K pneumoniae) Cas and Cascade proteins.    -   69. The vector of any one of paragraphs 1 to 29, wherein the Cas        and Cascade are E coli, C difficile, P aeruginosa, K pneumoniae,        P furiosus or B halodurans Cas and Cascade proteins.    -   70. The vector of any preceding paragraph, wherein the Cas3 is a        Cas3 of a CRISPR/Cas locus of a first bacterial or archaeal        species, wherein the distance between the Cas3-encoding sequence        of the locus and its cognate promoter is further than the        distance between the Cas3-encoding sequence and the respective        promoter comprised by the vector.    -   The cognate promoter here is the one that controls expression of        Cas3 in the wild-type locus.    -   71. The vector of any preceding paragraph, wherein the distance        between the promoter and the Cas3-encoding sequence and/or        Cascade protein-encoding sequence(s) is shorter than in a        corresponding wild-type Type I locus.    -   A corresponding locus is a wild-type locus of a bacterial or        archaeal species or strain that comprises an endogenous        CRISPR/Cas system encoding the Cas3 and/or Cascade proteins of        the type that are also encoded by the vector. Thus, when the        vector comprises an operon, the operon may comprise Cas3- and        Cascade-encoding nucleotide sequences that are not in a natural        configuration.    -   72. The vector of any preceding paragraph, wherein the vector        comprises (i) a CRISPR array for producing crRNAs in the host        cell and/or (ii) one or more nucleotide sequences encoding one        or more single guide RNAs (gRNAs), wherein the crRNAs or gRNAs        are cognate to the Cas3 (and optionally cognate to the Cascade        proteins).    -   73. The vector of paragraph 72 when dependent from paragraph 4,        wherein the array or gRNA-encoding sequence(s) are comprised by        the operon and under the control of the promoter.    -   74. The vector of paragraph 72, wherein the array or        gRNA-encoding sequence(s) are under the control of a promoter        that is different from the promoter that controls the expression        of the Cas3.    -   75. The vector of any one of paragraphs 72 to 74, wherein one or        more of the crRNAs or gRNAs comprises a spacer sequence that is        capable of hybridising to a target nucleotide sequence of the        host cell, wherein the target sequence is adjacent a PAM, the        PAM being cognate to the Cas3.    -   Thus, the spacer hybridises to the protospacer to guide the Cas3        to the protospacer. Optionally, the Cas3 cuts the protospacer,        eg, using exo- and/or endonuclease activity of the Cas3.        Optionally, the Cas3 removes a plurality (eg, at least 2, 3, 4,        5, 6, 7, 8, 9 or 10) nucleotides from the protospacer.    -   76. The vector of paragraph 75, wherein the target sequence is a        chromosomal sequence of the host cell.    -   77. The vector of paragraph 75 or 76, wherein the Cas3 is        operable to cut the target sequence.    -   78. The vector of any preceding paragraph, wherein the vector is        a plasmid or phagemid.    -   79. A delivery vehicle comprising the vector of any preceding        paragraph, wherein the delivery vehicle is capable of delivering        the vector into the host cell.    -   80. The vehicle of paragraph 79, wherein the delivery vehicle is        a phage, non-replicative transduction particle, nanoparticle        carrier, bacterium or liposome.    -   The phage or particles comprise phage coat proteins        encapsidating DNA, wherein the DNA comprises the vector.        Suitable examples of phage and particles are disclosed in U.S.        Ser. No. 15/985,658 (and its equivalent publication by USPTO)        the disclosures of which are incorporated herein by reference        for possible use in the invention and for providing one or more        features that may be included in the claims herein. Phage or        particle is capable of infecting the cell, thereby introducing        the vector into the cell.    -   81. The vector or vehicle of any preceding paragraph, wherein        the host cell is a bacterial or archaeal cell, optionally, the        host cell is a C difficile, P aeruginosa, K pneumoniae (eg,        carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum        Beta-Lactamase (ESBL)-producing K pneumoniae), E coli (eg,        ESBL-producing E. coli, or E. coli ST131-O25b:H4), H pylori, S        pneumoniae or S aureus cell.    -   82. The vector or vehicle of any preceding paragraph for        administration to a human or animal subject for treating or        reducing the risk of a disease or condition in the subject.    -   83. The vector or vehicle of paragraph 82, wherein the disease        or condition is an infection of the subject with host cells (eg,        bacterial cells), or wherein the disease or condition is        mediated by host cells (eg, bacterial cells).    -   84. A pharmaceutical composition comprising the vector or        vehicle of any preceding paragraph and a pharmaceutically        acceptable diluent, excipient or carrier.    -   85. A method of amplifying copies of a DNA encoding a functional        Cas protein (optionally a Cas nuclease) in a bacterial or        archaeal production strain of cells, the method comprising        -   (a) Providing production strain cells, each cell comprising            a copy of said DNA, wherein each DNA comprises a nucleotide            sequence encoding said Cas, wherein the nucleotide sequence            is under the control of a promoter for controlling the            expression of the Cas in the production strain cell, the DNA            comprising an origin of replication that is operable in the            cell for replication of the DNA;        -   (b) Culturing the cells to allow replication of the DNA,            whereby the DNA is amplified; and        -   (c) Optionally isolating copies of the DNA,    -   86. The method of paragraph 85, wherein the promoter is a        constitutive promoter.    -   87. The method of paragraph 85, wherein the promoter is        repressible (optionally repressible by a tetracycline repressor        or a lac repressor).    -   88. The method of paragraph 85, wherein the promoter is        inducible.    -   89. The method of any one of paragraphs 85 to 88, wherein the        promoter is a medium strength promoter.    -   90. The method of any one of paragraphs 85 to 89, wherein the        promoter has an Anderson Score (AS) of 0.5>AS>0.1.    -   91. The method of any one of paragraphs 85 to 90, wherein the        nucleotide sequence encoding said Cas is under the control of a        promoter and translation initiation site (TIS) combination that        is capable of producing expression of green fluorescent protein        (GFP) from a first expression operating unit (EOU) in E. coli        strain BW25113 cells with a fluorescence of from 0.5 to 4 times        the fluorescence produced in E. coli strain BW25113 cells using        a second EOU comprising a P10 promoter (SEQ ID NO: 1) combined        with a BCD14 TIS (SEQ ID NO: 2), wherein the EOUs differ only in        their promoter and TIS combinations, wherein each EOU comprises        (in 5′ to 3′ direction) an upstream initiator, the respective        promoter, the respective TIS, a nucleotide sequence encoding        GFP, a 3′ UTR, a transcription terminator and a downstream        insulator.    -   92. The method of paragraph 91, wherein fluorescence using the        first EOU is 0.5 to 2 times the fluorescence using the second        EOU.    -   93. The method of any one of paragraphs 85 to 92, wherein the        nuclease is Cas3 and optionally the DNA or cell encodes cognate        Cascade proteins and/or one or more crRNAs that are operable for        Cas nuclease targeting.    -   For example, the targeting is targeting of the Cas to a        protospacer sequence comprised by a host cell chromosome or an        episome thereof. In another example the targeting is in a        recombineering method and the Cas is targeted to a protospacer        sequence of a DNA that has been introduced into or amplified in        the host cell. In an example of such recombineering, the host        cell is an E coli cell.    -   94. The method of any one of paragraphs 85 to 92, wherein the        Cas is a Cas9.    -   95. The method of any one of paragraphs 85 to 92, wherein the        Cas is a Type IIIA csm protein or a Type IIIB cmr protein.    -   96. The method of any one of paragraphs 85 to 92, wherein the        Cas is a Csf1.    -   97. The method of any one of paragraphs 85 to 92, wherein the        Cas is a Cpf1.    -   98. The method of any one of paragraphs 85 to 92, wherein the        Cas is a Cas13 (optionally Cas13a or Cas13b).    -   99. The method of any one of paragraphs 85 to 92, wherein the        Cas is selected from a Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d,        Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10,        Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a,        Cas13b and Cas13c.    -   100. The method of any one of paragraphs 85 to 99, wherein the        production strain cells comprise a helper phage genome that is        inducible to produce phage coat proteins in the cells, wherein        the method further comprises inducing production of the phage        proteins and causing packaging of the amplified DNA into phage        particles or non-self-replicative transduction particles, and        further isolating the phage or transduction particles and        optionally formulating the particles into a pharmaceutical        composition for administration to a human or animal subject for        treating or reducing the risk of a disease or condition in the        subject.    -   101. The method of paragraph 100, wherein the particles are        capable of infecting target host cells in the subject and        transducing the cells with the DNA, wherein the Cas and crRNAs        (or gRNAs) encoded by the DNA are expressed in the cells, the        crRNAs or (gRNAs) being operable to guide the Cas to a target        nucleotide sequence (optionally a chromosomal sequence)        comprised by the cells, wherein the Cas cuts the target        sequences in the cells, thereby killing host cells and treating        or reducing the risk of the disease or condition.    -   102. The method of paragraph 101, wherein the host cells are        bacterial or archaeal cells, optionally, the host cells are C        difficile, P aeruginosa, K pneumoniae (eg, carbapenem-resistant        Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase        (ESBL)-producing K pneumoniae), E coli (eg, ESBL-producing E.        coli, or E. coli ST131-O25b:H4), H pylori, S pneumoniae or S        aureus cells.    -   103. The method of any one of paragraphs 85 to 102, wherein each        DNA is comprised by a high copy number vector, optionally a high        copy number plasmid (an optionally the promoter is a        constitutive promoter).    -   104. The method of any one of paragraphs 85 to 103, wherein each        DNA is comprised by a vector or vehicle according to any one of        paragraphs 1 to 83.    -   105. Use of an attenuated strength promoter in a DNA construct        comprising a nucleotide sequence encoding a functional Cas        protein (optionally a Cas nuclease) that is under the control of        the promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for enhancing        the yield of amplified DNA produced by the production host        cells.    -   Thus, said enhancing may be relative to the yield produced using        a strong promoter, eg, a strong constitutive promoter (for        example a promoter having an Anderson Score (AS) of AS≥0.5). In        another example, the strong promoter is a promoter comprised by        a promoter and translation initiation site (TIS) combination        that is capable of producing expression of green fluorescent        protein (GFP) from a first expression operating unit (EOU) in E.        coli strain BW25113 cells with a fluorescence of >4 times the        fluorescence produced in E. coli strain BW25113 cells using a        second EOU comprising a P10 promoter (SEQ ID NO: 1) combined        with a BCD14 TIS (SEQ ID NO: 2), wherein the EOUs differ only in        their promoter and TIS combinations, wherein each EOU comprises        (in 5′ to 3′ direction) an upstream initiator, the respective        promoter, the respective TIS, a nucleotide sequence encoding        GFP, a 3′ UTR, a transcription terminator and a downstream        insulator.    -   106. The use of paragraph 105, wherein the use is for enhancing        said yield by        -   (d) reducing toxicity of the Cas in the production strain;        -   (e) reducing mutation of the DNA (optionally the            Cas-encoding sequence) in the production strain;        -   (f) promoting production cell viability during the            amplification of the DNA; and/or        -   (g) reducing the occurrence of Cas cutting of DNA            (optionally cutting of production host cell chromosomal DNA            or said DNA construct).    -   107. Use of an attenuated strength promoter in a DNA construct        comprising a nucleotide sequence encoding a functional Cas        protein (optionally a Cas nuclease) that is under the control of        the promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for reducing        toxicity of the Cas in the production strain.    -   108. Use of an attenuated strength promoter in a DNA construct        comprising a nucleotide sequence encoding a functional Cas        protein (optionally a Cas nuclease) that is under the control of        the promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for reducing        mutation of the DNA (optionally the Cas-encoding sequence) in        the production strain.    -   109. Use of an attenuated strength promoter in a DNA construct        comprising a nucleotide sequence encoding a functional Cas        protein (optionally a Cas nuclease) that is under the control of        the promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for promoting        production cell viability during the amplification of the DNA.    -   110. Use of an attenuated strength promoter in a DNA construct        comprising a nucleotide sequence encoding a functional Cas        protein (optionally a Cas nuclease) that is under the control of        the promoter, in a method of amplifying copies of the DNA in a        population of bacterial or archaeal production strain cells, the        method comprising culturing the cells to allow replication of        the DNA thereby amplifying the DNA in the cells, for reducing        the occurrence of Cas cutting of DNA.    -   111. A method for enhancing the yield of amplified copies of a        DNA construct in a population of bacterial or archaeal        production strain cells, wherein the construct comprises a        nucleotide sequence encoding a functional Cas protein        (optionally a Cas nuclease) that is under the control of a        promoter, the method comprising culturing the cells to allow        replication of the DNA thereby amplifying the DNA in the cells,        wherein the promoter is an attenuated strength promoter.    -   112. A method for reducing toxicity of a functional Cas protein        (optionally a Cas nuclease) in a population of bacterial or        archaeal production strain cells in a process of amplifying        copies of a DNA construct, wherein the construct comprises a        nucleotide sequence encoding the Cas and the sequence is under        the control of a promoter, the method comprising culturing the        cells to allow replication of the DNA thereby amplifying the DNA        in the cells, wherein the promoter is an attenuated strength        promoter.    -   113. A method for reducing mutation of a DNA construct encoding        a functional Cas protein (optionally a Cas nuclease) in a        population of bacterial or archaeal production strain cells in a        process of amplifying copies of the construct, wherein the        construct comprises a nucleotide sequence encoding the Cas and        the sequence is under the control of a promoter, the method        comprising culturing the cells to allow replication of the DNA        thereby amplifying the DNA in the cells, wherein the promoter is        an attenuated strength promoter.    -   114. A method for promoting production cell viability of a        population of bacterial or archaeal production strain cells in a        process of amplifying copies of a DNA construct comprised by the        cells, wherein the construct comprises a nucleotide sequence        encoding a functional Cas protein (optionally a Cas nuclease)        and the sequence is under the control of a promoter, the method        comprising culturing the cells to allow replication of the DNA        thereby amplifying the DNA in the cells, wherein the promoter is        an attenuated strength promoter.    -   115. A method for reducing the occurrence of Cas nuclease        cutting of a DNA construct in a population of bacterial or        archaeal production strain cells in a process of amplifying        copies of the construct, wherein the construct comprises a        nucleotide sequence encoding the Cas and the sequence is under        the control of a promoter, the method comprising culturing the        cells to allow replication of the DNA thereby amplifying the DNA        in the cells, wherein the promoter is an attenuated strength        promoter.    -   116. The use of paragraph 108 or 110, or the method of paragraph        113 or 115, wherein the mutation or cutting is mutation or        cutting of host cell chromosomal DNA or the construct DNA.    -   117. The use or method of any one of paragraphs 105 to 116,        wherein the promoter is a constitutive promoter.    -   118. The use or method of any one of paragraphs 105 to 117,        wherein the promoter is repressible (optionally repressible by a        tetracycline repressor or a lac repressor).    -   In an example, the promoter is a constitutive promoter and        optionally the DNA is comprised by a high copy number plasmid or        phagemid.    -   119. The use or method of any one of paragraphs 105 to 118,        wherein the promoter is P_(LtetO-1), P_(LlacO-1) or a        repressible homologue thereof.    -   P_(LlacO-1) is repressed by lac repressor (LacR). P_(LtetO-1) is        repressed by tet repressor (TetR).    -   120. The use or method of any one of paragraphs 105 to 119,        wherein the promoter is a medium strength promoter.    -   121. The use or method of any one of paragraphs 105 to 120,        wherein the promoter has an Anderson Score (AS) of 0.5>AS>0.1.    -   122. The use or method of any one of paragraphs 105 to 121,        wherein the nucleotide sequence encoding said Cas is under the        control of a promoter and translation initiation site (TIS)        combination that is capable of producing expression of green        fluorescent protein (GFP) from a first expression operating unit        (EOU) in E. coli strain BW25113 cells with a fluorescence of        from 0.5 to 4 times the fluorescence produced in E. coli strain        BW25113 cells using a second EOU comprising a P10 promoter (SEQ        ID NO: 1) combined with a BCD14 TIS (SEQ ID NO: 2), wherein the        EOUs differ only in their promoter and TIS combinations, wherein        each EOU comprises (in 5′ to 3′ direction) an upstream        initiator, the respective promoter, the respective TIS, a        nucleotide sequence encoding GFP, a 3′ UTR, a transcription        terminator and a downstream insulator.    -   123. The use or method of paragraph 122, wherein fluorescence        using the first EOU is 0.5 to 2 times the fluorescence using the        second EOU.    -   124. The use or method of any one of paragraphs 105 to 123,        wherein the nuclease is Cas3 and optionally the DNA construct        encodes cognate Cascade proteins.    -   125. The use or method of any one of paragraphs 105 to 123,        wherein the Cas is a Cas9.    -   126. The use or method of any one of paragraphs 105 to 123,        wherein the Cas is a Type IIIA csm protein or a Type IIIB cmr        protein.    -   127. The use or method of any one of paragraphs 105 to 123,        wherein the Cas is a Csf1.    -   128. The use or method of any one of paragraphs 105 to 123,        wherein the Cas is a Cpf1.    -   129. The use or method of any one of paragraphs 105 to 123,        wherein the Cas is a Cas13 (optionally Cas13a or Cas13b).    -   130. The use or method of any one of paragraphs 105 to 123,        wherein the Cas is selected from a Cas3, Cas8a, Cas5, Cas8b,        Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10,        Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1,        C2c1, C2c3, Cas13a, Cas13b and Cas13c.    -   131. The use or method of any one of paragraphs 105 to 130,        wherein the DNA construct comprises one or more nucleotide        sequences for producing crRNAs or gRNAs that are operable for        Cas nuclease targeting.    -   132. The use or method of any one of paragraphs 105 to 131,        wherein the production strain cells comprise a helper phage        genome that is inducible to produce phage coat proteins in the        cells, wherein the method further comprises inducing production        of the phage proteins and causing packaging of the amplified DNA        into phage particles or non-self-replicative transduction        particles, and further isolating the phage or transduction        particles and optionally formulating the particles into a        pharmaceutical composition for administration to a human or        animal subject for treating or reducing the risk of a disease or        condition in the subject.    -   133. The method of paragraph 132, wherein the particles are        capable of infecting target host cells in the subject and        transducing the cells with the DNA, wherein the Cas and crRNAs        (or gRNAs) encoded by the DNA are expressed in the cells, the        crRNAs or (gRNAs) being operable to guide the Cas to a target        nucleotide sequence (optionally a chromosomal sequence)        comprised by the cells, wherein the Cas cuts the target        sequences in the cells, thereby killing host cells and treating        or reducing the risk of the disease or condition.    -   134. The method of paragraph 133, wherein the host cells are        bacterial or archaeal cells, optionally, the host cells are C        difficile, P aeruginosa, K pneumoniae (eg, carbapenem-resistant        Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase        (ESBL)-producing K pneumoniae), E coli (eg, ESBL-producing E.        coli, or E. coli ST131-O25b:H4), H pylori, S pneumoniae or S        aureus cells.    -   135. The use or method of any one of paragraphs 105 to 134,        wherein each DNA is comprised by a high copy number vector,        optionally a high copy number plasmid (an optionally the        promoter is a constitutive promoter).    -   136. The use or method of any one of paragraphs 105 to 135,        wherein each DNA is comprised by a vector according to any one        of paragraphs 1 to 78 and 81 to 83.

Clauses

The invention provides, by way of example, the following Clauses; thefeatures of these are combinable with any other disclosure herein.

-   -   1. A nucleic acid vector for introduction into a host cell, the        vector comprising a first nucleotide sequence encoding a Type I        Cas3 and a second nucleotide sequence encoding one or more        Cascade proteins, wherein the first and second sequences are        under the control of one or more promoters comprised by the        vector for expression of the proteins in the cell.    -   2. The vector of Clause 1, wherein the vector comprises an        operon for expression in the cell of the Cas3 and Cascade        proteins from a Cas module, the module comprising the nucleotide        sequences encoding the Cas3 and Cascade proteins, and the operon        comprising the Cas module under the control of a promoter for        controlling the expression of both the Cas3 and Cascade        proteins.    -   3. The vector of Clause 2, wherein        -   (a) the first sequence is between the promoter and the            second sequence in the operon;        -   (b) the operon comprises no Cas-encoding nucleotide            sequences between the promoter and the first nucleotide            sequence; and/or        -   (c) the operon comprises (in 5′ to 3′ direction) the            promoter, the first sequence and the second sequence.    -   4. The vector of any preceding Clause, wherein each promoter is        a constitutive promoter.    -   5. The vector of any one of Clauses 1 to 3, wherein the promoter        is repressible (optionally repressible by a tetracycline        repressor or lac repressor).    -   6. The vector of any one of Clauses 1 to 3, wherein the promoter        is inducible.    -   7. The vector of any preceding Clause, wherein the first        sequence is under the control of a medium strength promoter.    -   8. The vector of any preceding Clause, wherein the first        sequence is under the control of a promoter that has an Anderson        Score (AS) of 0.5>AS>0.1.    -   9. The vector of any preceding Clause, wherein the first        sequence (and optionally the second sequence) is under the        control of a promoter and translation initiation site (TIS)        combination that is capable of producing expression of green        fluorescent protein (GFP) from a first expression operating unit        (EOU) in E. coli strain BW25113 cells with a fluorescence of        from 0.5 to 4 times the fluorescence produced in E. coli strain        BW25113 cells using a second EOU comprising a P10 promoter (SEQ        ID NO: 1) combined with a BCD14 TIS (SEQ ID NO: 2), wherein the        EOUs differ only in their promoter and TIS combinations, wherein        each EOU comprises (in 5′ to 3′ direction) an upstream        initiator, the respective promoter, the respective TIS, a        nucleotide sequence encoding GFP, a 3′ UTR, a transcription        terminator and a downstream insulator.    -   10. The vector of Clause 9, wherein fluorescence using the first        EOU is 0.5 to 2 times the fluorescence using the second EOU.    -   11. The vector of any preceding Clause, wherein the vector        comprises an origin of replication that is operable in the host        cell.    -   12. The vector of any preceding Clause, wherein the vector        comprises an origin of replication that is operable in a        bacterial cell of a vector production strain, wherein the Cas3        is not operable in the production strain cell to target and cut        a chromosomal sequence thereof.    -   13. The vector of Clause 12, wherein the first sequence is under        the control of a promoter that is capable of controlling        expression of the Cas3 at a level that is not toxic to the        production strain cell.    -   14. The vector of any preceding Clause, wherein the vector is a        high copy number vector.    -   15. The vector of any preceding Clause, wherein the first        nucleotide sequence or operon is comprised by a mobile genetic        element.    -   16. The vector of any preceding Clause, wherein the vector is        devoid of a Cas adaption module.    -   17. The vector of any preceding Clause, wherein the vector is        devoid of nucleotide sequence encoding one, more or all of a        Cas1, Cas2, Cas4, Cas6, Cas7 and Cas 8.    -   18. The vector of any preceding Clause, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas11, Cas7 and Cas8a1.    -   19. The vector of Clause 18, wherein the vector comprises        nucleotide sequence encoding Cas3′ and/or Cas3″.    -   20. The vector or Clause 19, wherein the nucleotide sequences        encoding the Cas3′ and/or Cas3″ are between the promoter and the        sequence(s) recited in Clause 18.    -   21. The vector of any one of Clauses 18 to 20, wherein the host        cell comprises a Type IA CRISPR array that is cognate with the        Cas3.    -   22. The vector of any one of Clauses 18 to 20, wherein the host        cell comprises an endogenous Type IB, C, U, D, E or F CRISPR/Cas        system.    -   23. The vector of any one of Clauses 1 to 17, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas8b1, Cas7 and Cas5.    -   24. The vector of Clause 23, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in Clause 23.    -   25. The vector of Clause 23 or 24, wherein the host cell        comprises a Type IB CRISPR array that is cognate with the Cas3.    -   26. The vector of Clause 23 or 24, wherein the host cell        comprises an endogenous Type IA, C, U, D, E or F CRISPR/Cas        system.    -   27. The vector of any one of Clauses 1 to 17, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas5, Cas8c and Cas7.    -   28. The vector of Clause 27, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in Clause 27.    -   29. The vector of Clause 27 or 28, wherein the host cell        comprises a Type IC CRISPR array that is cognate with the Cas3.    -   30. The vector of Clause 27 or 28, wherein the host cell        comprises an endogenous Type IA, B, U, D, E or F CRISPR/Cas        system.    -   31. The vector of any one of Clauses 1 to 17, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas8U2, Cas7, Cas5 and Cas6.    -   32. The vector of Clause 31, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in Clause 31.    -   33. The vector of Clause 31 or 32, wherein the host cell        comprises a Type IU CRISPR array that is cognate with the Cas3.    -   34. The vector of Clause 31 or 32, wherein the host cell        comprises an endogenous Type IA, B, C, D, E or F CRISPR/Cas        system.    -   35. The vector of any one of Clauses 1 to 17, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas10d, Cas7 and Cas5.    -   36. The vector of Clause 35, wherein the vector comprises a        nucleotide sequence encoding Cas3′ and/or Cas3″.    -   37. The vector of Clause 36, wherein the nucleotide sequences        encoding the Cas3′ and/or Cas3″ are between the promoter and the        sequence(s) recited in Clause 35.    -   38. The vector of any one of Clauses 35 to 37, wherein the host        cell comprises a Type ID CRISPR array that is cognate with the        Cas3.    -   39. The vector of any one of Clauses 35 to 37, wherein the host        cell comprises an endogenous Type IA, B, C, U, E or F CRISPR/Cas        system.    -   40. The vector of any one of Clauses 1 to 17, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas8e, Cas11, Cas7, Cas5 and Cas6.    -   41. The vector of Clause 40, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in Clause 40.    -   42. The vector of Clause 40 or 41, wherein the host cell        comprises a Type IE CRISPR array that is cognate with the Cas3.    -   43. The vector of Clause 40 or 41, wherein the host cell        comprises an endogenous Type IA, B, C, D, U or F CRISPR/Cas        system.    -   44. The vector of any one of Clauses 1 to 17, wherein the vector        comprises (optionally in 5′ to 3′ direction) nucleotide sequence        encoding one, more or all of Cas8f, Cas5, Cas7 and Cas6f.    -   45. The vector of Clause 44, wherein the vector comprises a        nucleotide sequence encoding Cas3 between the promoter and the        sequence(s) recited in Clause 44, wherein the vector is devoid        of nucleotide sequence encoding further Cas between the promoter        and the sequence encoding the Cas3.    -   46. The vector of Clause 44 or 45, wherein the host cell        comprises a Type IF CRISPR array that is cognate with the Cas3.    -   47. The vector of Clause 44 or 45, wherein the host cell        comprises an endogenous Type IA, B, C, D, U or E CRISPR/Cas        system.    -   48. The vector of any one of Clauses 1 to 17, wherein the Cas        and Cascade are        -   (a) Type IA Cas and Cascade proteins;        -   (b) Type IB Cas and Cascade proteins;        -   (c) Type IC Cas and Cascade proteins;        -   (d) Type ID Cas and Cascade proteins;        -   (e) Type IE Cas and Cascade proteins;        -   (f) Type IF Cas and Cascade proteins; or        -   (g) Type IU Cas and Cascade proteins.    -   49. The vector of any preceding Clause, wherein the Cas and        Cascade are E coli (optionally Type IE or IF) Cas and Cascade        proteins.    -   50. The vector of Clause 49, wherein the E coli is        ESBL-producing E. coli or E. coli ST131-O25b:H4.    -   51. The vector of any preceding Clause, wherein the Cas and        Cascade are        -   (a) Clostridium (eg, C difficile) Cas and Cascade proteins,            optionally C difficile resistant to one or more antibiotics            selected from aminoglycosides, lincomycin, tetracyclines,            erythromycin, clindamycin, penicillins, cephalosporins and            fluoroquinolones;        -   (b) Pseudomonas aeruginosa Cas and Cascade proteins,            optionally P aeruginosa resistant to one or more antibiotics            selected from carbapenems, aminoglycosides, cefepime,            ceftazidime, fluoroquinolones, piperacillin and tazobactam;            or        -   (c) Klebsiella pneumoniae (eg, carbapenem-resistant            Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase            (ESBL)-producing K pneumoniae) Cas and Cascade proteins.    -   52. The vector of any preceding Clause, wherein the Cas and        Cascade are E coli, C difficile, P aeruginosa, K pneumoniae, P        furiosus or B halodurans Cas and Cascade proteins.    -   53. The vector of any preceding Clause, wherein the Cas3 is a        Cas3 of a CRISPR/Cas locus of a first bacterial or archaeal        species, wherein the distance between the Cas3-encoding sequence        of the locus and its cognate promoter is further than the        distance between the Cas3-encoding sequence and the respective        promoter comprised by the vector.    -   54. The vector of any preceding Clause, wherein the distance        between the promoter and the Cas3-encoding sequence and/or        Cascade protein-encoding sequence(s) is shorter than in a        corresponding wild-type Type I locus.    -   55. The vector of any preceding Clause, wherein the vector        comprises (i) a CRISPR array for producing crRNAs in the host        cell and/or (ii) one or more nucleotide sequences encoding one        or more guide RNAs (gRNAs or single gRNAs), wherein the crRNAs        or gRNAs are cognate to the Cas3 (and optionally cognate to the        Cascade proteins).    -   56. The vector of Clause 55 when dependent from Clause 2,        wherein the array or gRNA-encoding sequence(s) are comprised by        the operon and under the control of the promoter.    -   57. The vector of Clause 56, wherein the array or gRNA-encoding        sequence(s) are under the control of a promoter that is        different from the promoter that controls the expression of the        Cas3.    -   58. The vector of Clause 56 or 57, wherein one or more of the        crRNAs or gRNAs comprises a spacer sequence that is capable of        hybridising to a target nucleotide sequence of the host cell,        wherein the target sequence is adjacent a PAM, the PAM being        cognate to the Cas3.    -   59. The vector of Clause 58, wherein the target sequence is a        chromosomal sequence of the host cell.    -   60. The vector of Clause 58 or 59, wherein the Cas3 is operable        to cut the target sequence.    -   61. The vector of any preceding Clause, wherein the vector is a        plasmid or phagemid.    -   62. A delivery vehicle comprising the vector of any preceding        Clause, wherein the delivery vehicle is capable of delivering        the vector into the host cell.    -   63. The vehicle of Clause 62, wherein the delivery vehicle is a        phage, non-replicative transduction particle, nanoparticle        carrier, bacterium or liposome.    -   64. The vector or vehicle of any preceding Clause, wherein the        host cell is a bacterial or archaeal cell, optionally, the host        cell is a C difficile, P aeruginosa, K pneumoniae (eg,        carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum        Beta-Lactamase (ESBL)-producing K pneumoniae), E coli (eg,        ESBL-producing E. coli, or E. coli ST131-O25b:H4), H pylori, S        pneumoniae or S aureus cell.    -   65. The vector or vehicle of any preceding Clause for        administration to a human or animal subject for treating or        reducing the risk of a disease or condition in the subject.    -   66. The vector or vehicle of Clause 65, wherein the disease or        condition is an infection of the subject with host cells (eg,        bacterial cells), or wherein the disease or condition is        mediated by host cells (eg, bacterial cells).    -   67. A pharmaceutical composition comprising the vector or        vehicle of any preceding Clause and a pharmaceutically        acceptable diluent, excipient or carrier.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications and patent applications and all US equivalentpatent applications and patents are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference. Reference is made to WO2017/118598, US20180140698,US20170246221, US20180273940, US20160115488, US20180179547,US20170175142, US20160024510, US20150064138, US20170022499,US20160345578, US20180155729, US20180200342, WO2017112620, WO2018081502,PCT/EP2018/066954, PCT/EP2018/066980, PCT/EP2018/071454 and U.S. Ser.No. 15/985,658 and equivalent publications by the US Patent andTrademark Office (USPTO) or WIPO, the disclosures of which areincorporated herein by reference for providing disclosure that may beused in the present invention and/or to provide one or more features(eg, of a vector) that may be included in one or more claims herein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” or similar as used herein refers toall permutations and combinations of the listed items preceding theterm. For example, “A, B, C, or combinations thereof is intended toinclude at least one of: A, B, C, AB, AC, BC, or ABC, and if order isimportant in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC,or CAB. Continuing with this example, expressly included arecombinations that contain repeats of one or more item or term, such asBB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilledartisan will understand that typically there is no limit on the numberof items or terms in any combination, unless otherwise apparent from thecontext.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the followingnon-limiting Examples.

EXAMPLES

The examples illustrate fast and precision killing of Escherichia colistrains. As a model programmable nuclease system, we used a CRISPRguided vector (CGV™) to specifically target Escherichia coli MG1655.

Example 1 Single-Vector Cas3 & Cascade: Type I CRISPR-Cas SystemTargeting E. Coli

A plasmid (which we call a CRISPR Guided Vector™, CGV™) was constructedcomprising an operon with nucleotide sequences encoding a Type I Cas3and Cascade proteins under the control of a common promoter. C.difficile Type IB Cas3 and Cascade was used. A cognate CRISPR arraycomprising C. difficile repeat sequences and spacer sequence fortargeting an E. coli host cell chromosome was also introduced intotarget cells. An adaptation module containing Cas1, Cas2 and Cas4 wasomitted in the vector (see FIG. 1A). In the wild-type C. difficile TypeIB CRISPR/Cas locus, the cas3 gene is 3′ of the Cascade genes (cas8b1,cas7 and cas5) and thus spaced away from the promoter upstream of theCascade genes. When we tried this arrangement, we found killing of E.coli cells, but surprisingly when we changed to a synthetic operonarrangement (in 5′ to 3′ orientation) of promoter, cas3, cas8b1, cas7and cas5 we saw significantly higher killing of the target E. colicells.

Results using this synthetic operon arrangement are shown in FIGS.1A-1C. In FIG. 1B there is shown a dilution series (10¹-10⁶) of dropspots (5 μl) of target E. coli MG1655 cells harboring the CGV on LB agarplates with and without inducers. CRISPR/Cas induction surprisinglykilled 99.9% of the population (FIG. 1C, grey bar). Growth in absence ofinduction is shown in black. CGV™ refers to a CRISPR Guided Vector™,which is a nucleic acid vector comprising nucleotide sequences encodingCRISPR/Cas components.

We also managed to achieve desirable targeted killing of E coli cellsusing a similar set-up, except that E coli Type IE Cas and Cascade wereused, together with a cognate array targeting host cell E colichromosomal DNA (data not shown). In this case, a vector was usedcomprising (in 5′ to 3′ direction) a promoter controlling the expressionof Cas3, Cas8e, Cas 11, Cas7, Cas5 and Cas6 in an operon.

Materials and Methods

E. coli MG1655 was grown in lysogeny broth (LB) with shaking (250 rpm)at 37° C. When necessary, cultures were supplemented with tetracycline(10 μg/mL), and spectinomycin (400 μg/mL).

To construct a plasmid containing C. difficile CRISPR system underarabinose inducible pBAD promoter, cas3, cas6, cas8b, cas7 and cas5genes from C. difficile were amplified and cloned in a low copy numberplasmid (pSC101 ori). cas3 was located in the beginning of the operonfollowed by cas6, cas8b, cas7 and cas5. The adaptation module(consisting of cas1, cas2, and cas4) was omitted in the vector (FIG.1A). A second plasmid containing an IPTG inducible single-spacer arraytargeting a chromosomal intergenic region in E. coli MG1655 wasconstructed (FIG. 1A). The spacer was cloned under control of theIPTG-inducible Ptrc promoter, in a CloDF13 ori backbone. It contains 37nucleotides from the genome of E. coli MG1655(ctttgccgcgcgcttcgtcacgtaattctcgtcgcaa) (SEQ ID NO: 26). Additionally,the 3′-CCT protospacer adjacent motif (PAM) is located adjacent to theselected target sequence in the genome of E. coli MG1655 (FIG. 1A).

To perform killing assays, both plasmids were transformed into E. coliMG1655 by electroporation. Transformants were grown in liquid LB withantibiotics to mid-log phase, and the killing efficiency was determinedby serial dilution and spot plating onto LB, and LB+inducers (0.5 mMIPTG and 1% arabinose). Viability was calculated by counting colonyforming units (CFUs) on the plates and data were calculated as viablecell concentration (CFU/ml).

Example 2 Single-Vector Cas3-Cascade & Array: Type I CRISPR-Cas SystemTargeting E. Coli

A plasmid (which we call a CRISPR Guided Vector™, CGV™, which is anucleic acid vector comprising nucleotide sequences encoding CRISPR/Cascomponents) was constructed comprising an operon with nucleotidesequences encoding a Type I Cas3 and Cascade proteins under the controlof a common promoter. C. difficile Type IB Cas3 and Cascade was used.Adaptation module containing Cas1, Cas2 and Cas4 was omitted in thevector. A cognate CRISPR array comprising C. difficile repeat sequencesand spacer sequence for targeting an E. coli host cell chromosome wasalso cloned in the vector (see FIG. 2A). Similarly we also constructed aplasmid comprising of an operon with nucleotide sequences encoding E.coli Type IE Cas3 and Cascade proteins under control of a commonpromoter. The E. coli adaption module containing Cas1 and Cas2 wasomitted, in the vector. A cognate CRISPR array comprising E. coli repeatsequences and spacer sequence for targeting an E. coli host cellchromosome was also cloned in the vector.

The CGV containing the C. difficile CRISPR-Cas system was transformedinto E. coli MG1655 which contains a pks sequence incorporated into thegenome. Results using this synthetic operon arrangement are shown inFIGS. 2A-2C. In FIG. 2B there is shown a dilution series (10¹-10⁵) ofdrop spots (5 μl) of target E. coli MG1655 cells harboring the CGV onsynthetic medium (SM) agar plates with and without inducers. CRISPR/Casinduction resulted in more than 2-log₁₀ reductions in viable cells of E.coli MG1655 (FIG. 2C, grey bar). Growth in absence of induction is shownin black. CGV™ refers to a CRISPR Guided Vector™.

The survival of E. coli MG1655 upon induction was followed over time byplating the cultures in serial dilutions every 60 minutes, for 2 h (FIG.3A) Killing curves revealed that CRISPR/Cas induction mediated rapidkilling of E. coli MG1655, generating a two-log₁₀ reduction in E. coliby the first 60 minutes. FIG. 3B shows a dilution series (10¹-10⁶) ofdrop spots (5 μl) of induced and non-induced cultures of target E. coliMG1655 on SM agar plates.

The CGV containing the E. coli CRISPR-Cas system was transformed intoother E. coli MG1655 cells which contain a lambda sequence incorporatedinto the genome. Results using this synthetic operon arrangement areshown in FIGS. 6A-6B. In FIG. 6A there is shown a dilution series(10¹-10⁵) of drop spots (5 μl) of target E. coli MG1655 cells harboringthe CGV on synthetic medium (SM) agar plates with and without inducers.CRISPR/Cas induction resulted in more than 2-log₁₀ reductions in viablecells of E. coli MG1655 (FIG. 6B, grey bar). Growth in absence ofinduction is shown in black. In a repeat experiment (not shown) we saw a3-log₁₀ reductions in viable cells of E. coli MG1655 with CRISPR/Casinduction.

Materials and Methods

E. coli MG1655 was grown in synthetic medium (SM) with shaking (250 rpm)at 37° C. Cultures were supplemented with 10 μg/mL tetracycline whenrequired.

To construct a plasmid containing C. difficile CRISPR system underarabinose inducible pBAD promoter, cas3, cas6, cas8b, cas7 and cas5genes from C. difficile were amplified and cloned in a low copy numberplasmid (pSC101 ori). cas3 was located in the beginning of the operonfollowed by cas6, cas8b, cas7 and cas5. Additionally, an IPTG induciblesingle-spacer array targeting a chromosomal intergenic region in E. coliMG1655 was included in the vector under control of the IPTG-induciblePtrc promoter (FIG. 2A). It contains 37 nucleotides from the PKS gene(previously integrated into the genome of E. coli MG1655)(gtttggcgatggcgcgggtgtggttgtgcttcggcgt) (SEQ ID NO: 27). Additionally,the 3′-CCT protospacer adjacent motif (PAM) is located adjacent to theselected target sequence in the genome of E. coli MG1655 (FIG. 2A).

To construct a plasmid containing E. coli CRISPR system under arabinoseinducible pBAD promoter, cas3, cse1, cse2, cas7, cas5 and cas6 genesfrom E. coli were amplified and cloned in a low copy number plasmid(pSC101 ori). The operon comprised (in 5′ to 3′ direction) cas3 followedby cse1 cse2, cas7, cas5 and cas6. Additionally, an IPTG induciblesingle-spacer array targeting a chromosomal intergenic region in E. coliMG1655 was included in the vector under control of the IPTG-induciblePtrc promoter. It contained 32 nucleotides from the lambda sequence(previously integrated into the genome of E. coli MG1655)(tgggatgcctaccgcaagcagcttggcctgaa) (SEQ ID NO: 28) and found toefficiently target in Brouns et al., 2008 (Science. 2008 Aug. 15;321(5891):960-4. doi: 10.1126/science.1159689; “Small CRISPR RNAs guideantiviral defense in prokaryotes”). Additionally, the 3′-ATG protospaceradjacent motif (PAM) is located adjacent to the selected target sequencein the genome of E. coli MG1655.

The CGVs were transformed into E. coli MG1655 by electroporation.Transformants were grown in liquid SM with antibiotics to mid-log phase,and the killing efficiency was determined by serial dilution and spotplating onto LB, and LB+inducers (0.5 mM IPTG and 1% arabinose).Viability was calculated by counting colony forming units (CFUs) on theplates and data were calculated as viable cell concentration (CFU/ml).

To perform killing curves, E. coli MG1655 harboring the CGV was grown inliquid SM with antibiotics to mid-log phase. The culture was dividedinto two tubes and either inducers (0.5 mM IPTG and 1% arabinose) or PBSwere added. Survival of the strain was followed over time by plating thecultures in serial dilutions (10¹-10⁶) of drop spots (5 μl) every 60minutes, for 2 h, on SM plates with antibiotics. Survival frequency wascalculated by counting colony forming units (CFUs) on the plates anddata were calculated as viable cell concentration (CFU/ml).

Example 3 Precision Killing of Target Strain E. Coli MG1655 in aMicrobiome

An artificial microbial consortium was constructed to study theefficiency of the CGV carrying the CRISPR-Cas system of C. difficile, tospecifically target E. coli MG1655 in the presence of other microbes,mimicking the human microbiome.

The synthetic consortium consisted of three strains (two differentspecies) with differential antibiotic resistance profiles: astreptomycin-resistant E. coli MG1655 (target strain), anampicillin-resistant E. coli Top10, and a chloramphenicol-resistantLactococcus lactis NZ9000. To create the consortium, bacterial cultureswere grown separately in Brain Heart Infusion broth (BHI, optimal growthmedium for L. lactis) to mid-log phase and mixed in fresh BHI broth withand without inducers. After 1 h induction at 30° C., the composition ofthe consortium was determined by counting viable colonies on selectiveplates. Induction of the CRISPR system in the mixed community, resultedin >10-fold killing of target E. coli MG1655, while leaving E. coliTop10 and L. lactis NZ9000 cell populations unharmed (FIG. 4A). In FIG.4B there is shown a dilution series (10¹-10⁵) of drop spots (5 μl) ofthe synthetic consortium after 1 h induction on BHI agar plates.

Additionally, CRISPR killing of target strain E. coli MG1655 in thesynthetic microbial consortium was compared to a pure culture (ie,target strain E. coli MG1655 that is not mixed with another strain orspecies). Unexpectedly, in both conditions, killing of 3 logs wasachieved when plated on BHI agar plates with inducers (FIG. 5A). Thus,surprisingly the killing in the microbiome setting was as efficient asthe killing in pure culture. In FIG. 5B there is shown a dilution series(10¹-10⁵) of drop spots (5 μl) of the synthetic consortium and E. coliMG1655 in pure culture on BHI agar plates with and without inducers.

Materials and Methods

E. coli MG1655, E. coli Top10, and Lactococcus lactis NZ9000 were grownin BHI broth with shaking (250 rpm) at 30° C. Cultures were supplementedwith 1000 μg/mL streptomycin, 100 μg/mL ampicillin, or 10 μg/mLchloramphenicol, respectively.

To create the consortium, bacterial cultures were grown in BHI withappropriate antibiotics to mid-log phase. Cultures were washed twice inPBS to remove the antibiotics and mixed in fresh BHI broth. The mixedculture was spotted onto BHI plates with streptomycin, ampicillin orchloramphenicol to quantify the initial concentration of E. coli MG1655,E. coli Top10 and L. lactis NZ9000, respectively. The mixed culture wasdivided into two tubes and either inducers (0.5 mM IPTG and 1%arabinose) or PBS were added. After 1 h induction at 30° C., thecomposition of the consortium was calculated by counting colony formingunits (CFUs) on selective plates and data were calculated as viable cellconcentration (CFU/ml).

Example 4 Use of Promoter Repression in Vector Amplification Strains

We engineered an E coli Top10 production strain cell populationcomprising plasmid CGV DNA and an expressible sequence encoding a Tetrepressor (TetR). The DNA comprised a Cas9-encoding nucleotide sequenceunder the control of a Tet promoter (pLtetO-1 promoter). The promoter isnormally constitutively ON, but it was repressed by TetR in our cells.Thus, in this way we could successfully culture the cells and amplifythe CGV without observing adverse toxicity due to Cas9 expression.

In an experiment in the absence of repression, we did not observe anycolonies of production strain bacteria, and we surmise that this was dueto Cas9 toxicity. We believe, in addition to providing a way ofincreasing CGV yield (eg, for subsequent packaging into phage ornon-self-replicative transduction particles), our method usingrepression can minimize selection for mutations in the DNA that wouldotherwise be forced by higher Cas9 expression and cutting (eg, due toCGV cutting).

REFERENCES

-   Mutalik et al, Nat Methods. 2013 April; 10(4):354-60. doi:    10.1038/nmeth. 2404. Epub 2013 Mar. 10, “Precise and reliable gene    expression via standard transcription and translation initiation    elements”.

TABLE 1 Example Bacteria Optionally, the target host cells are cells ofa genus or species selected from this Table and/or the production straincells are cells of a genus or species selected from this TableAbiotrophia Acidocella Actinomyces Alkalilimnicola AquaspirillumAbiotrophia defectiva Acidocella aminolytica Actinomyces bovisAlkalilimnicola ehrlichii Aquaspirillum polymorphum AcaricomesAcidocella facilis Actinomyces denticolens Alkaliphilus AquaspirillumAcaricomes phytoseiuli Acidomonas Actinomyces europaeus Alkaliphilusoremlandii putridiconchylium Acetitomaculum Acidomonas methanolicaActinomyces georgiae Alkaliphilus transvaalensis Aquaspirillum serpensAcetitomaculum ruminis Acidothermus Actinomyces gerencseriaeAllochromatium Aquimarina Acetivibrio Acidothermus cellulolyticusActinomyces Allochromatium vinosum Aquimarina latercula Acetivibriocellulolyticus Acidovorax hordeovulneris Alloiococcus ArcanobacteriumAcetivibrio ethanolgignens Acidovorax anthurii Actinomyces howelliiAlloiococcus otitis Arcanobacterium Acetivibrio multivorans Acidovoraxcaeni Actinomyces hyovaginalis Allokutzneria haemolyticumAcetoanaerobium Acidovorax cattleyae Actinomyces israelii Allokutzneriaalbata Arcanobacterium pyogenes Acetoanaerobium noterae Acidovoraxcitrulli Actinomyces johnsonii Altererythrobacter Archangium AcetobacterAcidovorax defluvii Actinomyces meyeri Altererythrobacter ishigakiensisArchangium gephyra Acetobacter aceti Acidovorax delafieldii Actinomycesnaeslundii Altermonas Arcobacter Acetobacter cerevisiae Acidovoraxfacilis Actinomyces neuii Altermonas haloplanktis Arcobacter butzleriAcetobacter cibinongensis Acidovorax konjaci Actinomyces odontolyticusAltermonas macleodii Arcobacter cryaerophilus Acetobacter estunensisAcidovorax temperans Actinomyces oris Alysiella Arcobacter halophilusAcetobacter fabarum Acidovorax valerianellae Actinomyces radingaeAlysiella crassa Arcobacter nitrofigilis Acetobacter ghanensisAcinetobacter Actinomyces slackii Alysiella filiformis Arcobacterskirrowii Acetobacter indonesiensis Acinetobacter baumannii Actinomycesturicensis Aminobacter Arhodomonas Acetobacter lovaniensis Acinetobacterbaylyi Actinomyces viscosus Aminobacter aganoensis Arhodomonas aquaeoleiAcetobacter malorum Acinetobacter bouvetii Actinoplanes Aminobacteraminovorans Arsenophonus Acetobacter nitrogenifigens Acinetobactercalcoaceticus Actinoplanes auranticolor Aminobacter niigataensisArsenophonus nasoniae Acetobacter oeni Acinetobacter gerneriActinoplanes brasiliensis Aminobacterium Acetobacter orientalisAcinetobacter haemolyticus Actinoplanes consettensis Aminobacteriummobile Arthrobacter Acetobacter orleanensis Acinetobacter johnsoniiActinoplanes deccanensis Aminomonas Arthrobacter agilis Acetobacterpasteurianus Acinetobacter junii Actinoplanes derwentensis Aminomonaspaucivorans Arthrobacter albus Acetobacter pornorurn Acinetobacterlwoffi Actinoplanes digitatis Ammoniphilus Arthrobacter aurescensAcetobacter senegalensis Acinetobacter parvus Actinoplanes durhamensisAmmoniphilus oxalaticus Arthrobacter chlorophenolicus Acetobacterxylinus Acinetobacter radioresistens Actinoplanes ferrugineusAmmoniphilus oxalivorans Arthrobacter citreus AcetobacteriumAcinetobacter schindleri Actinoplanes globisporus AmphibacillusArthrobacter crystallopoietes Acetobacterium bakii Acinetobacter soliActinoplanes humidus Amphibacillus xylanus Arthrobacter cumminsiiAcetobacterium carbinolicum Acinetobacter tandoii Actinoplanes italicusAmphritea Arthrobacter globiformis Acetobacterium dehalogenansAcinetobacter tjernbergiae Actinoplanes liguriensis Amphritea balenaeArthrobacter Acetobacterium fimetarium Acinetobacter towneriActinoplanes lobatus Amphritea japonica histidinolovorans Acetobacteriummalicum Acinetobacter ursingii Actinoplanes missouriensis AmycolatopsisArthrobacter ilicis Acetobacterium paludosum Acinetobacter venetianusActinoplanes palleronii Amycolatopsis alba Arthrobacter luteusAcetobacterium tundrae Acrocarpospora Actinoplanes philippinensisAmycolatopsis albidoflavus Arthrobacter methylotrophus Acetobacteriumwieringae Acrocarpospora corrugata Actinoplanes rectilineatusAmycolatopsis azurea Arthrobacter mysorens Acetobacterium woodiiAcrocarpospora Actinoplanes regularis Amycolatopsis coloradensisArthrobacter nicotianae Acetofilamentum macrocephala ActinoplanesAmycolatopsis lurida Arthrobacter nicotinovorans Acetofilamentum rigidumAcrocarpospora pleiomorpha teichomyceticus Amycolatopsis mediterraneiArthrobacter oxydans Acetohalobium Actibacter Actinoplanes utahensisAmycolatopsis rifamycinica Arthrobacter pascens Acetohalobium arabaticumActibacter sediminis Actinopolyspora Amycolatopsis rubida ArthrobacterAcetomicrobium Actinoalloteichus Actinopolyspora halophila Amycolatopsissulphurea phenanthrenivorans Acetomicrobium faecale ActinoalloteichusActinopolyspora mortivallis Amycolatopsis tolypomycina ArthrobacterAcetomicrobium flavidum cyanogriseus Actinosynnema Anabaenapolychromogenes Acetonema Actinoalloteichus Actinosynnema mirum Anabaenacylindrica Atrhrobacter protophormiae Acetonema longum hymeniacidonisActinotalea Anabaena flos-aquae Arthrobacter AcetothermusActinoalloteichus spitiensis Actinotalea fermentans Anabaena variabilispsychrolactophilus Acetothermus paucivorans Actinobaccillus AerococcusAnaeroarcus Arthrobacter ramosus Acholeplasma Actinobacillus capsulatusAerococcus sanguinicola Anaeroarcus burkinensis Arthrobactersulfonivorans Acholeplasma axanthum Actinobacillus delphinicolaAerococcus urinae Anaerobaculum Arthrobacter sulfureus Acholeplasmabrassicae Actinobacillus hominis Aerococcus urinaeequi Anaerobaculummobile Arthrobacter uratoxydans Acholeplasma cavigenitaliumActinobacillus indolicus Aerococcus urinaehominis AnaerobiospirillumArthrobacter ureafaciens Acholeplasma equifetale Actinobacilluslignieresii Aerococcus viridans Anaerobiospirillum Arthrobacter viscosusAcholeplasma granularum Actinobacillus minor Aeromicrobiumsucciniciproducens Arthrobacter woluwensis Acholeplasma hippikonActinobacillus muris Aeromicrobium erythreum Anaerobiospirillum thomasiiAsaia Acholeplasma laidlawii Actinobacillus Aeromonas Anaerococcus Asaiabogorensis Acholeplasma modicum pleuropneumoniae Aeromonas Anaerococcushydrogenalis Asanoa Acholeplasma morum Actinobacillus porcinusallosaccharophila Anaerococcus lactolyticus Asanoa ferrugineaAcholeplasma multilocale Actinobacillus rossii Aeromonas bestiarumAnaerococcus prevotii Asticcacaulis Acholeplasma oculi Actinobacillusscotiae Aeromonas caviae Anaerococcus tetradius Asticcacaulisbiprosthecium Acholeplasma palmae Actinobacillus seminis Aeromonasencheleia Anaerococcus vaginalis Asticcacaulis excentricus Acholeplasmaparvum Actinobacillus succinogenes Aeromonas Anaerofustis AtopobacterAcholeplasma pleciae Actinobaccillus suis enteropelogenes Anaerofustisstercorihominis Atopobacter phocae Acholeplasma vituli Actinobacillusureae Aeromonas eucrenophila Anaeromusa Atopobium AchromobacterActinobaculum Aeromonas ichthiosmia Anaeromusa acidaminophila Atopobiumfossor Achromobacter denitrificans Actinobaculum massiliense Aeromonasjandaei Anaeromyxobacter Atopobium minutum Achromobacter insolitusActinobaculum schaalii Aeromonas media Anaeromyxobacter Atopobiumparvulum Achromobacter piechaudii Actinobaculum suis Aeromonas popoffiidehalogenans Atopobium rimae Achromobacter ruhlandii Actinomyces urinaleAeromonas sobria Anaerorhabdus Atopobium vaginae Achromobacter spaniusActinocatenispora Aeromonas veronii Anaerorhabdus furcosa AureobacteriumAcidaminobacter Actinocatenispora rupis Agrobacterium AnaerosinusAureobacterium barkeri Acidaminobacter Actinocatenispora AgrobacteriumAnaerosinus glycerini Aurobacterium hydrogenoformans thailandicagelatinovorum Anaerovirgula Aurobacterium liquefaciens AcidaminococcusActinocatenispora sera Agrococcus Anaerovirgula multivorans AvibacteriumAcidaminococcus fermentans Actinocorallia Agrococcus citreusAncalomicrobium Avibacterium avium Acidaminococcus intestiniActinocorallia aurantiaca Agrococcus jenensis Ancalomicrobium adetumAvibacterium gallinarum Acidicaldus Actinocorallia aurea AgromonasAncylobacter Avibacterium paragallinarum Acidicaldus organivoransActinocorallia cavernae Agromonas oligotrophica Ancylobacter aquaticusAvibacterium volantium Acidimicrobium Actinocorallia glomerata AgromycesAneurinibacillus Azoarcus Acidimicrobium ferrooxidans Actinocoralliaherbida Agromyces fucosus Aneurinibacillus aneurinilyticus Azoarcusindigens Acidiphilium Actinocorallia libanotica Agromyces hippuratusAneurinibacillus migulanus Azoarcus tolulyticus Acidiphilium acidophilumActinocorallia longicatena Agromyces luteolus Aneurinibacillus Azoarcustoluvorans Acidiphilium angustum Actinomadura Agromyces mediolanusthermoaerophilus Azohydromonas Acidiphilium cryptum Actinomadura albaAgromyces ramosus Angiococcus Azohydromonas australica Acidiphiliummultivorum Actinomadura atramentaria Agromyces rhizospherae Angiococcusdisciformis Azohydromonas lata Acidiphilium organovorum ActinomaduraAkkermansia Angulomicrobium Azomonas Acidiphilium rubrum bangladeshensisAkkermansia muciniphila Angulomicrobium tetraedrale Azomonas agilisAcidisoma Actinomadura catellatispora Albidiferax Anoxybacillus Azomonasinsignis Acidisoma sibiricum Actinomadura chibensis Albidiferaxferrireducens Anoxybacillus pushchinoensis Azomonas macrocytogenesAcidisoma tundrae Actinomadura chokoriensis Albidovulum AquabacteriumAzorhizobium Acidisphaera Actinomadura citrea Albidovulum inexpectatumAquabacterium commune Azorhizobium caulinodans Acidisphaera rubrifaciensActinomadura coerulea Alcaligenes Aquabacterium parvum AzorhizophilusAcidithiobacillus Actinomadura echinospora Alcaligenes denitrificansAzorhizophilus paspali Acidithiobacillus albertensis Actinomadurafibrosa Alcaligenes faecalis Azospirillum Acidithiobacillus caldusActinomadura formosensis Alcanivorax Azospirillum brasilenseAcidithiobacillus ferrooxidans Actinomadura hibisca Alcanivoraxborkumensis Azospirillum halopraeferens Acidithiobacillus thiooxidansActinomadura kijaniata Alcanivorax jadensis Azospirillum irakenseAcidobacterium Actinomadura latina Algicola Azotobacter Acidobacteriumcapsulatum Actinomadura livida Algicola bacteriolytica Azotobacterbeijerinckii Actinomadura Alicyclobacillus Azotobacter chroococcumluteofluorescens Alicyclobacillus Azotobacter nigricans Actinomaduramacra disulfidooxidans Azotobacter salinestris Actinomadura maduraeAlicyclobacillus Azotobacter vinelandii Actinomadura oligosporasendaiensis Actinomadura pelletieri Alicyclobacillus vulcanalisActinomadura rubrobrunea Alishewanella Actinomadura rugatobisporaAlishewanella fetalis Actinomadura umbrina Alkalibacillus ActinomaduraAlkalibacillus verrucosospora haloalkaliphilus Actinomadura vinaceaActinomadura viridilutea Actinomadura viridis Actinomadura yumaensisBacillus Bacteroides Bibersteinia Borrelia Brevinema [see below]Bacteroides caccae Bibersteinia trehalosi Borrelia afzelii Brevinemaandersonii Bacteroides coagulans Bifidobacterium Borrelia americanaBrevundimonas Bacteriovorax Bacteroides eggerthii Bifidobacteriumadolescentis Borrelia burgdorferi Brevundimonas alba Bacteriovoraxstolpii Bacteroides fragilis Bifidobacterium angulatum Borreliacarolinensis Brevundimonas aurantiaca Bacteroides galacturonicusBifidobacterium animalis Borrelia coriaceae Brevundimonas diminutaBacteroides helcogenes Bifidobacterium asteroides Borrelia gariniiBrevundimonas intermedia Bacteroides ovatus Bifidobacterium bifidumBorrelia japonica Brevundimonas subvibrioides Bacteroides pectinophilusBifidobacterium boum Bosea Brevundimonas vancanneytii Bacteroidespyogenes Bifidobacterium breve Bosea minatitlanensis Brevundimonasvariabilis Bacteroides salyersiae Bifidobacterium catenulatum Boseathiooxidans Brevundimonas vesicularis Bacteroides stercorisBifidobacterium choerinum Brachybacterium Brochothrix Bacteroides suisBifidobacterium coryneforme Brachybacterium Brochothrix campestrisBacteroides tectus Bifidobacterium cuniculi alimentarium Brochothrixthermosphacta Bacteroides thetaiotaomicron Bifidobacterium dentiumBrachybacterium faecium Brucella Bacteroides uniformis Bifidobacteriumgallicum Brachybacterium Brucella canis Bacteroides ureolyticusBifidobacterium gallinarum paraconglomeratum Brucella neotomaeBacteroides vulgatus Bifidobacterium indicum Brachybacterium rhamnosumBryobacter Balnearium Bifidobacterium longum Brachybacterium Bryobacteraggregatus Balnearium lithotrophicum Bifidobacterium tyrofermentansBurkholderia Balneatrix magnumBifidobacterium Brachyspira Burkholderiaambifaria Balneatrix alpica merycicum Brachyspira alvinipulliBurkholderia andropogonis Balneola Bifidobacterium minimum Brachyspirahyodysenteriae Burkholderia anthina Balneola vulgaris BifidobacteriumBrachyspira innocens Burkholderia caledonica Barnesiellapseudocatenulatum Brachyspira murdochii Burkholderia caryophylliBarnesiella viscericola Bifidobacterium Brachyspira pilosicoliBurkholderia cenocepacia Bartonella pseudoIongum Burkholderia cepaciaBartonella alsatica Bifidobacterium pullorum Bradyrhizobium Burkholderiacocovenenans Bartonella bacilliformis Bifidobacterium ruminantiumBradyrhizobium canariense Burkholderia dolosa Bartonella clarridgeiaeBifidobacterium saeculare Bradyrhizobium elkanii Burkholderia fungorumBartonella doshiae Bifidobacterium subtile Bradyrhizobium japonicumBurkholderia glathei Bartonella elizabethae BifidobacteriumBradyrhizobium liaoningense Burkholderia glumae Bartonella grahamiithermophilum Brenneria Burkholderia graminis Bartonella henselaeBilophila Brenneria alni Burkholderia kururiensis Bartonella rochalimaeBilophila wadsworthia Brenneria nigrifluens Burkholderia multivoransBartonella vinsonii Biostraticola Brenneria quercina Burkholderiaphenazinium Bavariicoccus Biostraticola tofi Brenneria quercinaBurkholderia plantarii Bavariicoccus seileri Bizionia Brenneria salicisBurkholderia pyrrocinia Bdellovibrio Bizionia argentinensisBrevibacillus Burkholderia silvatlantica Bdellovibrio bacteriovorusBlastobacter Brevibacillus agri Burkholderia stabilis Bdellovibrioexovorus Blastobacter capsulatus Brevibacillus borstelensis Burkholderiathailandensis Beggiatoa Blastobacter denitrificans Brevibacillus brevisBurkholderia tropica Beggiatoa alba Blastococcus Brevibacilluscentrosporus Burkholderia unamae Beijerinckia Blastococcus aggregatusBrevibacillus choshinensis Burkholderia vietnamiensis Beijerinckiaderxii Blastococcus saxobsidens Brevibacillus invocatus ButtiauxellaBeijerinckia fluminensis Blastochloris Brevibacillus laterosporusButtiauxella agrestis Beijerinckia indica Blastochloris viridisBrevibacillus parabrevis Buttiauxella brennerae Beijerinckia mobilisBlastomonas Brevibacillus reuszeri Buttiauxella ferragutiae BelliellaBlastomonas natatoria Brevibacterium Buttiauxella gaviniae Belliellabaltica Blastopirellula Brevibacterium abidum Buttiauxella izardiiBellilinea Blastopirellula marina Brevibacterium album Buttiauxellanoackiae Bellilinea caldifistulae Blautia Brevibacterium aurantiacumButtiauxella warmboldiae Belnapia Blautia coccoides Brevibacteriumcelere Butyrivibrio Belnapia moabensis Blautia hansenii Brevibacteriumepidermidis Butyrivibrio fibrisolvens Bergeriella Blautia productaBrevibacterium Butyrivibrio hungatei Bergeriella denitrificans Blautiawexlerae frigoritolerans Butyrivibrio proteoclasticus BeutenbergiaBogoriella Brevibacterium halotolerans Beutenbergia cavernae Bogoriellacaseilytica Brevibacterium iodinum Bordetella Brevibacterium linensBordetella avium Brevibacterium lyticum Bordetella bronchisepticaBrevibacterium mcbrellneri Bordetella hinzii Brevibacterium otitidisBordetella holmesii Brevibacterium oxydans Bordetella parapertussisBrevibacterium paucivorans Bordetella pertussis Brevibacterium stationisBordetella petrii Bordetella trematum Bacillus B. acidiceler B.aminovorans B. glucanolyticus B. taeanensis B. lautus B. acidicola B.amylolyticus B. gordonae B. tequilensis B. lehensis B. acidiproducens B.andreesenii B. gottheilii B. thermantarcticus B. lentimorbus B.acidocaldarius B. aneurinilyticus B. graminis B. thermoaerophilus B.lentus B. acidoterrestris B. anthracis B. halmapalus B.thermoamylovorans B. licheniformis B. aeolius B. aquimaris B.haloalkaliphilus B. thermocatenulatus B. ligniniphilus B. aerius B.arenosi B. halochares B. thermocloacae B. litoralis B. aerophilus B.arseniciselenatis B. halodenitrificans B. thermocopriae B. locisalis B.agaradhaerens B. arsenicus B. halodurans B. thermodenitrificans B.luciferensis B. agri B. aurantiacus B. halophilus B. thermoglucosidasiusB. luteolus B. aidingensis B. arvi B. halosaccharovorans B. thermolactisB. luteus B. akibai B. aryabhattai B. hemicellulosilyticus B.thermoleovorans B. macauensis B. alcalophilus B. asahii B. hemicentrotiB. thermophilus B. macerans B. algicola B. atrophaeus B.herbersteinensis B. thermoruber B. macquariensis B. alginolyticus B.axarquiensis B. horikoshii B. thermosphaericus B. macyae B.alkalidiazotrophicus B. azotofixans B. horneckiae B. thiaminolyticus B.malacitensis B. alkalinitrilicus B. azotoformans B. horti B. thioparansB. mannanilyticus B. alkalisediminis B. badius B. huizhouensis B.thuringiensis B. marisflavi B. alkalitelluris B. barbaricus B. humi B.tianshenii B. marismortui B. altitudinis B. bataviensis B. hwajinpoensisB. trypoxylicola B. marmarensis B. alveayuensis B. beijingensis B.idriensis B. tusciae B. massiliensis B. alvei B. benzoevorans B. indicusB. validus B. megaterium B. amyloliquefaciens B. beringensis B. infantisB. vallismortis B. mesonae B. B. berkeleyi B. infernus B. vedderi B.methanolicus a. subsp. amyloliquefaciens B. beveridgei B. insolitus B.velezensis B. methylotrophicus B. a. subsp. plantarum B. bogoriensis B.invictae B. vietnamensis B. migulanus B. boroniphilus B. iranensis B.vireti B. mojavensis B. dipsosauri B. borstelensis B. isabeliae B.vulcani B. mucilaginosus B. drentensis B. brevis Migula B. isronensis B.wakoensis B. muralis B. edaphicus B. butanolivorans B. jeotgali B.weihenstephanensis B. murimartini B. ehimensis B. canaveralius B.kaustophilus B. xiamenensis B. mycoides B. eiseniae B. carboniphilus B.kobensis B. xiaoxiensis B. naganoensis B. enclensis B. cecembensis B.kochii B. zhanjiangensis B. nanhaiensis B. endophyticus B.cellulosilyticus B. kokeshiiformis B. peoriae B. nanhaiisediminis B.endoradicis B. centrosporus B. koreensis B. persepolensis B. nealsoniiB. farraginis B. cereus B. korlensis B. persicus B. neidei B.fastidiosus B. chagannorensis B. kribbensis B. pervagus B. neizhouensisB. fengqiuensis B. chitinolyticus B. krulwichiae B. plakortidis B.niabensis B. firmus B. chondroitinus B. laevolacticus B. pocheonensis B.niacini B. flexus B. choshinensis B. larvae B. polygoni B. novalis B.foraminis B. chungangensis B. laterosporus B. polymyxa B.oceanisediminis B. fordii B. cibi B. salexigens B. popilliae B. odysseyiB. formosus B. circulans B. saliphilus B. pseudalcalophilus B. okhensisB. fortis B. clarkii B. schlegelii B. pseudofirmus B. okuhidensis B.fumarioli B. clausii B. sediminis B. pseudomycoides B. oleronius B.funiculus B. coagulans B. selenatarsenatis B. psychrodurans B.oryzaecorticis B. fusiformis B. coahuilensis B. selenitireducens B.psychrophilus B. oshimensis B. galactophilus B. cohnii B. seohaeanensisB. psychrosaccharolyticus B. pabuli B. galactosidilyticus B. composti B.shacheensis B. psychrotolerans B. pakistanensis B. galliciensis B.curdlanolyticus B. shackletonii B. pulvifaciens B. pallidus B. gelatiniB. cycloheptanicus B. siamensis B. pumilus B. pallidus B. gibsonii B.cytotoxicus B. silvestris B. purgationiresistens B. panacisoli B.ginsengi B. daliensis B. simplex B. pycnus B. panaciterrae B.ginsengihumi B. decisifrondis B. siralis B. qingdaonensis B.pantothenticus B. ginsengisoli B. decolorationis B. smithii B.qingshengii B. parabrevis B. globisporus (eg, B. B. deserti B. soli B.reuszeri B. paraflexus g. subsp. Globisporus; or B. B. solimangrovi B.rhizosphaerae B. pasteurii g. subsp. Marinus) B. solisalsi B. rigui B.patagoniensis B. songklensis B. ruris B. sonorensis B. safensis B.sphaericus B. salarius B. sporothermodurans B. stearothermophilus B.stratosphericus B. subterraneus B. subtilis (eg, B. s. subsp.Inaquosorum, or B. s. subsp. Spizizenr, or B. s. subsp. Subtilis)Caenimonas Campylobacter Cardiobacterium Catenuloplanes CurtobacteriumCaenimonas koreensis Campylobacter coli Cardiobacterium hominisCatenuloplanes atrovinosus Curtobacterium albidum CaldalkalibacillusCampylobacter concisus Carnimonas Catenuloplanes castaneusCurtobacterium citreus Caldalkalibacillus uzonensis Campylobacter curvusCarnimonas nigrificans Catenuloplanes crispus CaldanaerobacterCampylobacter fetus Carnobacterium Catenuloplanes indicusCaldanaerobacter subterraneus Campylobacter gracilis Carnobacteriumalterfunditum Catenuloplanes japonicus Caldanaerobius Campylobacterhelveticus Carnobacterium divergens Catenuloplanes nepalensisCaldanaerobius fijiensis Campylobacter hominis Carnobacterium funditumCatenuloplanes niger Caldanaerobius Campylobacter hyointestinalisCarnobacterium gallinarum Chryseobacterium polysaccharolyticusCampylobacter jejuni Carnobacterium Chryseobacterium Caldanaerobius zeaeCampylobacter lari maltaromaticum balustinum CaldanaerovirgaCampylobacter mucosalis Carnobacterium mobile CitrobacterCaldanaerovirga acetigignens Campylobacter rectus Carnobacteriumviridans C. amalonaticus Caldicellulosiruptor Campylobacter showaeCaryophanon C. braakii Caldicellulosiruptor bescii Campylobactersputorum Caryophanon latum C. diversus Caldicellulosiruptorkristjanssonii Campylobacter upsaliensis Caryophanon tenue C. farmeriCaldicellulosiruptor owensensis Capnocytophaga Catellatospora C.freundii Capnocytophaga canimorsus Catellatospora citrea C. gilleniiCapnocytophaga cynodegmi Catellatospora C. koseri Capnocytophagagingivalis methionotrophica C. murliniae Capnocytophaga granulosaCatenococcus C. pasteurii ^([1]) Capnocytophaga haemolytica Catenococcusthiocycli C. rodentium Capnocytophaga ochracea C. sedlakiiCapnocytophaga sputigena C. werkmanii C. youngae Clostridium (see below)Coccochloris Coccochloris elabens Corynebacterium Corynebacteriumflavescens Corynebacterium variabile Clostridium Clostridium absonum,Clostridium aceticum, Clostridium acetireducens, Clostridiumacetobutylicum, Clostridium acidisoli, Clostridium aciditolerans,Clostridium acidurici, Clostridium aerotolerans, Clostridium aestuarii,Clostridium akagii, Clostridium aldenense, Clostridium aldrichii,Clostridium algidicarni, Clostridium algidixylanolyticum, Clostridiumalgifaecis, Clostridium algoriphilum, Clostridium alkalicellulosi,Clostridium aminophilum, Clostridium aminovalericum, Clostridiumamygdalinum, Clostridium amylolyticum, Clostridium arbusti, Clostridiumarcticum, Clostridium argentinense, Clostridium asparagiforme,Clostridium aurantibutyricum, Clostridium autoethanogenum, Clostridiumbaratii, Clostridium barkeri, Clostridium bartlettii, Clostridiumbeijerinckii, Clostridium bifermentans, Clostridium bolteae, Clostridiumbornimense, Clostridium botulinum, Clostridium bowmanii, Clostridiumbryantii, Clostridium butyricum, Clostridium cadaveris, Clostridiumcaenicola, Clostridium caminithermale, Clostridium carboxidivorans,Clostridium carnis, Clostridium cavendishii, Clostridium celatum,Clostridium celerecrescens, Clostridium cellobioparum, Clostridiumcellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi,Clostridium cellulovorans, Clostridium chartatabidum, Clostridiumchauvoei, Clostridium chromiireducens, Clostridium citroniae,Clostridium clariflavum, Clostridium clostridioforme, Clostridiumcoccoides, Clostridium cochlearium, Clostridium colletant, Clostridiumcolicanis, Clostridium colinum, Clostridium collagenovorans, Clostridiumcylindrosporum, Clostridium difficile, Clostridium diolis, Clostridiumdisporicum, Clostridium drakei, Clostridium durum, Clostridiumestertheticum, Clostridium estertheticum estertheticum, Clostridiumestertheticum laramiense, Clostridium fallax, Clostridium felsineum,Clostridium fervidum, Clostridium fimetarium, Clostridiumformicaceticum, Clostridium frigidicarnis, Clostridium frigoris,Clostridium ganghwense, Clostridium gasigenes, Clostridium ghonii,Clostridium glycolicum, Clostridium glycyrrhizinilyticum, Clostridiumgrantii, Clostridium haemolyticum, Clostridium halophilum, Clostridiumhastiforme, Clostridium hathewayi, Clostridium herbivorans, Clostridiumhiranonis, Clostridium histolyticum, Clostridium homopropionicum,Clostridium huakuii, Clostridium hungatei, Clostridium hydrogeniformans,Clostridium hydroxybenzoicum, Clostridium hylemonae, Clostridiumjejuense, Clostridium indolis, Clostridium innocuum, Clostridiumintestinale, Clostridium irregulare, Clostridium isatidis, Clostridiumjosui, Clostridium kluyveri, Clostridium lactatifermentans, Clostridiumlacusfryxellense, Clostridium laramiense, Clostridium lavalense,Clostridium lentocellum, Clostridium lentoputrescens, Clostridiumleptum, Clostridium limosum, Clostridium litorale, Clostridiumlituseburense, Clostridium ljungdahlii, Clostridium lortetii,Clostridium lundense, Clostridium magnum, Clostridium malenominatum,Clostridium mangenotii, Clostridium mayombei, Clostridiummethoxybenzovorans, Clostridium methylpentosum, Clostridiumneopropionicum, Clostridium nexile, Clostridium nitrophenolicum,Clostridium novyi, Clostridium oceanicum, Clostridium orbiscindens,Clostridium oroticum, Clostridium oxalicum, Clostridium papyrosolvens,Clostridium paradoxum, Clostridium paraperfringens (Alias: C. welchii),Clostridium paraputrificum, Clostridium pascui, Clostridiumpasteurianum, Clostridium peptidivorans, Clostridium perenne,Clostridium perfringens, Clostridium pfennigii, Clostridiumphytofermentans, Clostridium piliforme, Clostridium polysaccharolyticum,Clostridium populeti, Clostridium propionicum, Clostridiumproteoclasticum, Clostridium proteolyticum, Clostridium psychrophilum,Clostridium puniceum, Clostridium purinilyticum, Clostridiumputrefaciens, Clostridium putrificum, Clostridium quercicolum,Clostridium quinii, Clostridium ramosum, Clostridium rectum, Clostridiumroseum, Clostridium saccharobutylicum, Clostridium saccharogumia,Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum,Clostridium sardiniense, Clostridium sartagoforme, Clostridiumscatologenes, Clostridium schirmacherense, Clostridium scindens,Clostridium septicum, Clostridium sordellii, Clostridium sphenoides,Clostridium spiroforme, Clostridium sporogenes, Clostridiumsporosphaeroides, Clostridium stercorarium, Clostridium stercorariumleptospartum, Clostridium stercorarium stercorarium, Clostridiumstercorarium thermolacticum, Clostridium sticklandii, Clostridiumstraminisolvens, Clostridium subterminale, Clostridium sufflavum,Clostridium sulfidigenes, Clostridium symbiosum, Clostridium tagluense,Clostridium tepidiprofundi, Clostridium termitidis, Clostridium tertium,Clostridium tetani, Clostridium tetanomorphum, Clostridiumthermaceticum, Clostridium thermautotrophicum, Clostridiumthermoalcaliphilum, Clostridium thermobutyricum, Clostridiumthermocellum, Clostridium thermocopriae, Clostridiumthermohydrosulfuricum, Clostridium thermolacticum, Clostridiumthermopalmarium, Clostridium thermopapyrolyticum, Clostridiumthermosaccharolyticum, Clostridium thermosuccinogenes, Clostridiumthermosulfurigenes, Clostridium thiosulfatireducens, Clostridiumtyrobutyricum, Clostridium uliginosum, Clostridium ultunense,Clostridium villosum, Clostridium vincentii, Clostridium viride,Clostridium xylanolyticum, Clostridium xylanovorans DactylosporangiumDeinococcus Delftia Echinicola Dactylosporangium aurantiacum Deinococcusaerius Delftia acidovorans Echinicola pacifica Dactylosporangium fulvumDeinococcus apachensis Desulfovibrio Echinicola vietnamensisDactylosporangium matsuzakiense Deinococcus aquaticus Desulfovibriodesulfuricans Dactylosporangium roseum Deinococcus aquatilis DiplococcusDactylosporangium thailandense Deinococcus caeni Diplococcus pneumoniaeDactylosporangium vinaceum Deinococcus radiodurans Deinococcusradiophilus Enterobacter Enterobacter kobei FaecalibacteriumFlavobacterium E. aerogenes E. ludwigii Faecalibacterium prausnitziiFlavobacterium antarcticum E. amnigemis E. mori Fangia Flavobacteriumaquatile E. agglomerans E. nimipressuralis Fangia hongkongensisFlavobacterium aquidurense E. arachidis E. oryzae FastidiosipilaFlavobacterium balustinum E. asburiae E. pulveris Fastidiosipilasanguinis Flavobacterium croceum E. cancerogenous E. pyrinusFusobacterium Flavobacterium cucumis E. cloacae E. radicincitansFusobacterium nucleatum Flavobacterium daejeonense E. cowanii E.taylorae Flavobacterium defluvii E. dissolvens E. turicensisFlavobacterium degerlachei E. gergoviae E. sakazakii Enterobacter soliFlavobacterium E. helveticus Enterococcus denitrificans E. hormaecheiEnterococcus durans Flavobacterium filum E. intermedins Enterococcusfaecalis Flavobacterium flevense Enterococcus faecium Flavobacteriumfrigidarium Erwinia Flavobacterium mizutaii Erwinia haponticiFlavobacterium Escherichia okeanokoites Escherichia coli GaetbulibacterHaemophilus Ideonella Janibacter Gaetbulibacter saemankumensisElaemophilus aegyptius Ideonella azotifigens Janibacter anophelisGallibacterium Elaemophilus aphrophilus Idiomarina Janibactercorallicola Gallibacterium anatis Haemophilus felis Idiomarina abyssalisJanibacter limosus Gallicola Haemophilus gallinarum Idiomarina balticaJanibacter melonis Gallicola barnesae Haemophilus haemolyticusIdiomarina fontislapidosi Janibacter terrae Garciella Haemophilusinfluenzae Idiomarina loihiensis Jannaschia Garciella nitratireducensHaemophilus paracuniculus Idiomarina ramblicola Jannaschia cystaugensGeobacillus Haemophilus parahaemolyticus Idiomarina seosinensisJannaschia helgolandensis Geobacillus thermoglucosidasius Haemophilusparainfluenzae Idiomarina zobellii Jannaschia pohangensis Geobacillusstearothermophilus Haemophilus Ignatzschineria Jannaschia rubraGeobacter paraphrohaemolyticus Ignatzschineria larvae Geobacterbemidjiensis Haemophilus parasuis Janthinobacterium Geobacter bremensisHaemophilus pittmaniae Ignavigranum Janthinobacterium Geobacterchapellei Hafnia Ignavigranum ruoffiae agaricidamnosum Geobactergrbiciae Hafnia alvei Ilumatobacter Janthinobacterium lividum Geobacterhydrogenophilus Hahella Ilumatobacter fluminis Jejuia Geobacter lovleyiHahella ganghwensis Ilyobacter Jejuia pallidilutea Geobactermetallireducens Halalkalibacillus Ilyobacter delafieldiiJeotgalibacillus Geobacter pelophilus Halalkalibacillus halophilusIlyobacter insuetus Jeotgalibacillus Geobacter pickeringii HelicobacterIlyobacter polytropus alimentarius Geobacter sulfurreducens Helicobacterpylori Ilyobacter tartaricus Jeotgalicoccus GeodermatophilusJeotgalicoccus halotolerans Geodermatophilus obscurus GluconacetobacterGluconacetobacter xylinus Gordonia Gordonia rubripertincta KaistiaLabedella Listeria ivanovii Micrococcus Nesterenkonia Kaistia adipataLabedella gwakjiensis L. marthii Micrococcus luteus Nesterenkoniaholobia Kaistia soli Labrenzia L. monocytogenes Micrococcus lylaeNocardia Kangiella Labrenzia aggregata L. newyorkensis MoraxellaNocardia argentinensis Kangiella aquimarina Labrenzia alba L. ripariaMoraxella bovis Nocardia corallina Kangiella koreensis Labrenziaalexandrii L. rocourtiae Moraxella nonliquefaciens Nocardia Labrenziamarina L. seeligeri Moraxella osloensis otitidiscaviarum KerstersiaLabrys L. weihenstephanensis Nakamurella Kerstersia gyiorum Labrysmethylaminiphilus L. welshimeri Nakamurella multipartita KiloniellaLabrys miyagiensis Listonella Nannocystis Kiloniella laminariae Labrysmonachus Listonella anguillarum Nannocystis pusilia Klebsiella Labrysokinawensis Macrococcus Natranaerobius K. gramilomatis Labrysportucalensis Macrococcus bovicus Natranaerobius K. oxytoca Marinobacterthermophilus K. pneumoniae Lactobacillus Marinobacter algicolaNatranaerobius trueperi K. terrigena [see below] Marinobacter bryozoorumNaxibacter K. variicola Laceyella Marinobacter flavimaris Naxibacteralkalitolerans Kluyvera Laceyella putida Meiothermus Neisseria Kluyveraascorbata Lechevalieria Meiothermus ruber Neisseria cinerea KocuriaLechevalieria aerocolonigenes Methylophilus Neisseria denitrificansKocuria roasea Legionella Methylophilus methylotrophus Neisseriagonorrhoeae Kocuria varians [see below] Microbacterium Neisserialactamica Kurthia Listeria Microbacterium Neisseria mucosa Kurthiazopfii L. aquatica ammoniaphilum Neisseria sicca L. booriaeMicrobacterium arborescens Neisseria subflava L. cornellensisMicrobacterium liquefaciens Neptunomonas L. fleischmannii Microbacteriumoxydans Neptunomonas japonica L. floridensis L. grandensis L. grayi L.innocua Lactobacillus L. acetotolerans L. catenaformis L. mali L.parakefiri L. sakei L. acidifarinae L. ceti L. manihotivorans L.paralimentarius L. salivarius L. acidipiscis L. coleohominis L.mindensis L. paraplantarum L. sanfranciscensis L. acidophilus L.collinoides L. mucosae L. pentosus L. satsumensis Lactobacillus agilisL. composti L. murinus L. perolens L. secaliphilus L. algidus L.concavus L. nagelii L. plantarum L. sharpeae L. alimentarius L.coryniformis L. namurensis L. pontis L. siliginis L. amylolyticus L.crispatus L. nantensis L. protectus L. spicheri L. amylophilus L.crustorum L. oligofermentans L. psittaci L. suebicus L. amylotrophicusL. curvatus L. oris L. rennini L. thailandensis L. amylovorus L.delbrueckii subsp. bulgaricus L. panis L. reuteri L. ultunensis L.animalis L. delbrueckii subsp. L. pantheris L. rhamnosus L.vaccinostercus L. antri delbrueckii L. parabrevis L. rimae L. vaginalisL. apodemi L. delbrueckii subsp. lactis L. parabuchneri L. rogosae L.versmoldensis L. aviarius L. dextrinicus L. paracasei L. rossiae L. viniL. bifermentans L. diolivorans L. paracollinoides L. ruminis L.vitulinus L. brevis L. equi L. parafarraginis L. saerimneri L. zeae L.buchneri L. equigenerosi L. homohiochii L. jensenii L. zymae L.camelliae L. farraginis L. iners L. johnsonii L. gastricus L. casei L.farciminis L. ingluviei L. kalixensis L. ghanensis L. kitasatonis L.fermentum L. intestinalis L. kefiranofaciens L. graminis L. kunkeei L.fornicalis L. fuchuensis L. kefiri L. hammesii L. leichmannii L.fructivorans L. gallinarum L. kimchii L. hamsteri L. lindneri L.frumenti L. gasseri L. helveticus L. harbinensis L. malefermentans L.hilgardii L. hayakitensis Legionella Legionella adelaidensis Legionelladrancourtii Candidatus Legionella jeonii Legionella quinlivaniiLegionella anisa Legionella dresdenensis Legionella jordanis Legionellarowbothamii Legionella beliardensis Legionella drozanskii Legionellalansingensis Legionella rubrilucens Legionella birminghamensisLegionella dumoffii Legionella londiniensis Legionella sainthelensiLegionella bozemanae Legionella erythra Legionella longbeachaeLegionella santicrucis Legionella brunensis Legionella fairfieldensisLegionella lytica Legionella shakespearei Legionella busanensisLegionella fallonii Legionella maceachernii Legionella spiritensisLegionella cardiaca Legionella feeleii Legionella massiliensisLegionella steelei Legionella cherrii Legionella geestiana Legionellamicdadei Legionella steigerwaltii Legionella cincinnatiensis Legionellagenomospecies Legionella monrovica Legionella taurinensis Legionellaclemsonensis Legionella gormanii Legionella moravica Legionellatucsonensis Legionella donaldsonii Legionella gratiana Legionellanagasakiensis Legionella tunisiensis Legionella gresilensis Legionellanautarum Legionella wadsworthii Legionella hackeliae Legionellanorrlandica Legionella waltersii Legionella impletisoli Legionellaoakridgensis Legionella worsleiensis Legionella israelensis Legionellaparisiensis Legionella yabuuchiae Legionella jamestowniensis Legionellapittsburghensis Legionella pneumophila Legionella quateirensisOceanibulbus Paenibacillus Prevotella Quadrisphaera Oceanibulbusindolifex Paenibacillus thiaminolyticus Prevotella albensisQuadrisphaera granulorum Oceanicaulis Pantoea Prevotella amniiQuatrionicoccus Oceanicaulis alexandrii Pantoea agglomerans Prevotellabergensis Quatrionicoccus Oceanicola Prevotella bivia australiensisOceanicola batsensis Paracoccus Prevotella brevis Oceanicola granulosusParacoccus alcaliphilus Prevotella bryantii Quinella Oceanicolananhaiensis Paucimonas Prevotella buccae Quinella ovalis OceanimonasPaucimonas lemoignei Prevotella buccalis Oceanimonas baumanniiPectobacterium Prevotella copri Ralstonia OceaniserpentillaPectobacterium aroidearum Prevotella dentalis Ralstonia eutrophaOceaniserpentilla haliotis Pectobacterium atrosepticum Prevotelladenticola Ralstonia insidiosa Oceanisphaera Pectobacteriumbetavasculorum Prevotella disiens Ralstonia mannitolilyticaOceanisphaera donghaensis Pectobacterium cacticida Prevotella histicolaRalstonia pickettii Oceanisphaera litoralis Pectobacterium carnegieanaPrevotella intermedia Ralstonia Oceanithermus Pectobacterium carotovorumPrevotella maculosa pseudosolanacearum Oceanithermus desulfuransPectobacterium chrysanthemi Prevotella marshii Ralstonia syzygiiOceanithermus profundus Pectobacterium cypripedii Prevotellamelaninogenica Ralstonia solanacearum Oceanobacillus Pectobacteriumrhapontici Prevotella micans Ramlibacter Oceanobacillus caeniPectobacterium wasabiae Prevotella multiformis Ramlibacter henchirensisOceanospirillum Pianococcus Prevotella nigrescens Ramlibactertataouinensis Oceanospirillum linum Pianococcus citreus Prevotellaoralis Planomicrobium Prevotella oris Raoultella Planomicrobiumokeanokoites Prevotella oulorum Raoultella ornithinolytica PlesiomonasPrevotella pallens Raoultella planticola Plesiomonas shigelloidesPrevotella salivae Raoultella terrigena Proteus Prevotella stercoreaRathayibacter Proteus vulgaris Prevotella tannerae Rathayibacter caricisPrevotella timonensis Rathayibacter festucae Prevotella veroralisRathayibacter iranicus Providencia Rathayibacter rathayi Providenciastuartii Rathayibacter toxicus Pseudomonas Rathayibacter triticiPseudomonas aeruginosa Rhodobacter Pseudomonas alcaligenes Rhodobactersphaeroides Pseudomonas anguillispetica Ruegeria Pseudomonas fluorescensRuegeria gelatinovorans Pseudoalteromonas haloplanktis Pseudomonasmendocina Pseudomonas pseudoalcaligenes Pseudomonas putida Pseudomonastutzeri Pseudomonas syringae Psychrobacter Psychrobacter faecalisPsychrobacter phenylpyruvicus Saccharococcus Sagittula SanguibacterStenotrophomonas Tatlockia Saccharococcus thermophilus Sagittulastellata Sanguibacter keddieii Stenotrophomonas Tatlockia maceacherniiSaccharomonospora Salegentibacter Sanguibacter suarezii maltophiliaTatlockia micdadei Saccharomonospora azurea Salegentibacter salegensSaprospira Streptococcus Tenacibaculum Saccharomonospora cyaneaSalimicrobium Saprospira grandis Tenacibaculum Saccharomonospora viridisSalimicrobium album Sarcina [also see below] amylolyticum SaccharophagusSalinibacter Sarcina maxima Streptomyces Tenacibaculum discolorSaccharophagus degradans Salinibacter ruber Sarcina ventriculiStreptomyces Tenacibaculum Saccharopolyspora Salinicoccus Sebaldellaachromogenes gallaicum Saccharopolyspora erythraea Salinicoccusalkaliphilus Sebaldella termitidis Streptomyces cesalbus TenacibaculumSaccharopolyspora gregorii Salinicoccus hispanicus Streptomycescescaepitosus lutimaris Saccharopolyspora hirsuta Salinicoccus roseusSerratia Streptomyces cesdiastaticus Tenacibaculum Saccharopolysporahordei Salinispora Serratia fonticola Streptomyces cesexfoliatusmesophilum Saccharopolyspora rectivirgula Salinispora arenicola Serratiamarcescens Streptomyces fimbriatus Tenacibaculum Saccharopolysporaspinosa Salinispora tropica Sphaerotilus Streptomyces fradiaeskagerrakense Saccharopolyspora taberi Salinivibrio Sphaerotilus natansStreptomyces fulvissimus Tepidanaerobacter Saccharothrix Salinivibriocosticola Sphingobacterium Streptomyces griseoruber TepidanaerobacterSaccharothrix australiensis Salmonella Sphingobacterium multivorumStreptomyces griseus syntrophicus Saccharothrix coeruleofusca Salmonellabongori Staphylococcus Streptomyces lavendulae TepidibacterSaccharothrix espanaensis Salmonella enterica [see below] StreptomycesTepidibacter Saccharothrix longispora Salmonella subterraneaphaeochromogenes formicigenes Saccharothrix mutabilis Salmonella typhiStreptomyces Tepidibacter thalassicus Saccharothrix syringaethermodiastaticus Thermus Saccharothrix tangerinus Streptomycestubercidicus Thermus aquaticus Saccharothrix texasensis Thermusfiliformis Thermus thermophilus Staphylococcus S. arlettae S. equorum S.microti S. schleiferi S. agnetis S. felis S. muscae S. sciuri S. aureusS. fleurettii S. nepalensis S. simiae S. auricularis S. gallinarum S.pasteuri S. simulans S. capitis S. haemolyticus S. petrasii S.stepanovicii S. caprae S. hominis S. pettenkoferi S. succinus S.carnosus S. hyicus S. piscifermentans S. vitulinus S. caseolyticus S.intermedius S. pseudintermedius S. warneri S. chromogenes S. kloosii S.pseudolugdunensis S. xylosus S. cohnii S. leei S. pulvereri S.condimenti S. lentus S. rostri S. delphini S. lugdunensis S.saccharolyticus S. devriesei S. lutrae S. saprophyticus S. epidermidisS. lyticans S. massiliensis Streptococcus Streptococcus agalactiaeStreptococcus infantarius Streptococcus orisratti Streptococcusthermophilus Streptococcus anginosus Streptococcus iniae Streptococcusparasanguinis Streptococcus sanguinis Streptococcus bovis Streptococcusintermedius Streptococcus peroris Streptococcus sobrinus Streptococcuscanis Streptococcus lactarius Streptococcus pneumoniae Streptococcussuis Streptococcus constellatus Streptococcus milleri StreptococcusStreptococcus uberis Streptococcus downei Streptococcus mitispseudopneumoniae Streptococcus vestibularis Streptococcus dysgalactiaeStreptococcus mutans Streptococcus pyogenes Streptococcus viridansStreptococcus equines Streptococcus oralis Streptococcus rattiStreptococcus Streptococcus faecalis Streptococcus tigurinusStreptococcus salivariu zooepidemicus Streptococcus ferusUliginosibacterium Vagococcus Vibrio Virgibacillus XanthobacterVagococcus carniphilus Vibrio aerogenes Virgibacillus Xanthobacteragilis Uliginosibacterium gangwonense Vagococcus elongatus Vibrioaestuarianus halodenitrificans Xanthobacter Ulvibacter Vagococcus fessusVibrio albensis Virgibacillus aminoxidans Ulvibacter litoralisVagococcus fluvialis Vibrio alginolyticus pantothenticus XanthobacterUmezawaea Vagococcus lutrae Vibrio campbellii Weissella autotrophicusUmezawaea tangerina Vagococcus salmoninarum Vibrio cholerae Weissellacibaria Xanthobacter flavus Undibacterium Variovorax Vibriocincinnatiensis Weissella confusa Xanthobacter tagetidis Undibacteriumpigrum Variovorax boronicumulans Vibrio coralliilyticus Weissellahalotolerans Xanthobacter viscosus Ureaplasma Variovorax dokdonensisVibrio cyclitrophicus Weissella hellenica Xanthomonas Ureaplasmaurealyticum Variovorax paradoxus Vibrio diazotrophicus Weissellakandleri Xanthomonas Variovorax soli Vibrio fluvialis Weissellakoreensis albilineans Ureibacillus Veillonella Vibrio furnissiiWeissella minor Xanthomonas alfalfae Ureibacillus composti Veillonellaatypica Vibrio gazogenes Weissella Xanthomonas Ureibacillus suwonensisVeillonella caviae Vibrio halioticoli paramesenteroides arboricolaUreibacillus terrenus Veillonella criceti Vibrio harveyi Weissella soliXanthomonas Ureibacillus thermophilus Veillonella dispar Vibrioichthyoenteri Weissella thailandensis axonopodis Ureibacillusthermosphaericus Veillonella montpellierensis Vibrio mediterraneiWeissella viridescens Xanthomonas Veillonella parvula Vibriometschnikovii Williamsia campestris Veillonella ratti Vibrio mytiliWilliamsia marianensis Xanthomonas citri Veillonella rodentium Vibrionatriegens Williamsia maris Xanthomonas codiaei Venenivibrio Vibrionavarrensis Williamsia serinedens Xanthomonas Venenivibriostagnispumantis Vibrio nereis Winogradskyella cucurbitae Vibrionigripulchritudo Winogradskyella Xanthomonas Verminephrobacter Vibrioordalii thalassocola euvesicatoria Verminephrobacter eiseniae Vibrioorientalis Wolbachia Xanthomonas fragariae Vibrio parahaemolyticusWolbachia persica Xanthomonas fuscans Verrucomicrobium Vibriopectenicida Xanthomonas gardneri Verrucomicrobium spinosum Vibriopenaeicida Wolinella Xanthomonas hortorum Vibrio proteolyticus Wolinellasuccinogenes Xanthomonas hyacinthi Vibrio shilonii Xanthomonas perforansVibrio splendidus Zobellia Xanthomonas phaseoli Vibrio tubiashiiZobellia galactanivorans Xanthomonas pisi Vibrio vulnificus Zobelliauliginosa Xanthomonas populi Zoogloea Xanthomonas theicola Zoogloearamigera Xanthomonas Zoogloea resiniphila translucens Xanthomonasvesicatoria Xylella Xylella fastidiosa Xylophilus Xylophilus ampelinusXenophilus Yangia Yersinia mollaretii Zooshikella Zobellella Xenophilusazovorans Yangia pacifica Yersinia philomiragia Zooshikella ganghwensisZobellella denitrificans Xenorhabdus Yaniella Yersinia pestisZunongwangia Zobellella taiwanensis Xenorhabdus beddingii Yaniella flavaYersinia pseudotuberculosis Zunongwangia profunda Xenorhabdus bovieniiYaniella halotolerans Yersinia rohdei Zymobacter ZeaxanthinibacterXenorhabdus cabanillasii Yeosuana Yersinia ruckeri Zymobacter palmaeZeaxanthinibacter Xenorhabdus doucetiae Yeosuana aromativorans YokenellaZymomonas enoshimensis Xenorhabdus griffiniae Yersinia Yokenellaregensburgei Zymomonas mobilis Zhihengliuella Xenorhabdus hominickiiYersinia aldovae Yonghaparkia Zymophilus Zhihengliuella Xenorhabduskoppenhoeferi Yersinia bercovieri Yonghaparkia alkaliphila Zymophiluspaucivorans halotolerans Xenorhabdus nematophila Yersinia enterocoliticaZavarzinia Zymophilus raffinosivorans Xylanibacterium Xenorhabduspoinarii Yersinia entomophaga Zavarzinia compransoris Xylanibacteriumulmi Xylanibacter Yersinia frederiksenii Xylanibacter oryzae Yersiniaintermedia Yersinia kristensenii

TABLE 2 Sequences Nucleic acid sequences herein are written in 5′ to3′ direction; amino acid sequences are written inN- to C-terminal direction. SEQ ID NO: 1 (P10)TTTCAATTTAATCATCCGGCTCGTATAATGTGTGGA SEQ ID NO: 2 (BCD14)GGGCCCAAGTTCACTTAAAAAGGAGATCAACAATGAAAGCAATTTTCGTACTGAAACATCTTAATCATGCGGTGGAGGGTTTCTAATG SEQ ID NO: 3 (gfp)ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCSEQ IDs NO: 4 & 29 (example Expression Operating Unit, EOU)The EOU is (in 5′ to 3′ direction):-[SEQ ID NO: 4]-[promoter]-[TIS]-[GFP-encoding nucleotide sequence]-[SEQ ID NO: 29] Where SEQ ID NO: 4 isGAATTCAAAAGATCTTAAGTAAGTAAGAGTATACGTATATCGGCTAATAACGTATGAAGGCGCTTCGGCGCCTTTTTTTATGGGGGTATTTTCATCCCAATCCACACGTCCAACGCACAGCAAACACCACGTCGACCCTATCAGCTGCGTGCTTTCTATGAGTCGTTGCTGCATAACTTGACAATTAATCATCCGGCTCG TATAATGTGTGGAASEQ ID NO: 29 is GGATCCAAACTCGAGTAAGGATCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTT ATASEQ ID NO: 5 (Example Shine Dalgarno Sequence) AAAGAGGAGAAASEQ ID NO: 26 (Spacer sequence) CTTTGCCGCGCGCTTCGTCACGTAATTCTCGTCGCAASEQ ID NO: 27 (Spacer sequence) GTTTGGCGATGGCGCGGGTGTGGTTGTGCTTCGGCGTSEQ ID NO: 28 (Spacer sequence) TGGGATGCCTACCGCAAGCAGCTTGGCCTGAA

TABLE 3 Anderson Promoter Collection SEQ ID Measured NO: IdentifierSequence^(a) Strength^(b)  6 BBa J23119TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC n/a  7 BBa J23100TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC 1  8 BBa J23101TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC 0.7  9 BBa J23102TTGACAGCTAGCTCAGTCCTAGGTACTGTGCTAGC 0.86 10 BBa J23103CTGATAGCTAGCTCAGTCCTAGGGATTATGCTAGC 0.01 11 BBa J23104TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGC 0.72 12 BBa J23105TTTACGGCTAGCTCAGTCCTAGGTACTATGCTAGC 0.24 13 BBa J23106TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC 0.47 14 BBa J23107TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGC 0.36 15 BBa J23108CTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC 0.51 16 BBa J23109TTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGC 0.04 17 BBa J23110TTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGC 0.33 18 BBa J23111TTGACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC 0.58 19 BBa J23112CTGATAGCTAGCTCAGTCCTAGGGATTATGCTAGC 0 20 BBa J23113CTGATGGCTAGCTCAGTCCTAGGGATTATGCTAGC 0.01 21 BBa J23114TTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGC 0.1 22 BBa J23115TTTATAGCTAGCTCAGCCCTTGGTACAATGCTAGC 0.15 23 BBa J23116TTGACAGCTAGCTCAGTCCTAGGGACTATGCTAGC 0.16 24 BBa J23117TTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAGC 0.06 25 BBa J23118TTGACGGCTAGCTCAGTCCTAGGTATTGTGCTAGC 0.56 aalso shown in the AndersonCatalog, see parts.igem.org/Promoters/Catalog/Anderson ^(b)Strength isthe Anderson Score (AS), e.g., a strength of 1 is a AS of 1. Reportedactivities of the promoters are given as the relative fluorescence ofplasmids in strain TG1 grown in LB media to saturation. A suitableplasmid is EX-Ptet-S-rbsRFP-P ″RFP reporter″ as described atparts.igem.org/Part:BBa_J61002; insertion of a promoter element betweenXbaI and SpeI sites results in a RFP reporter.

The invention claimed is:
 1. A production strain bacterial cellcomprising a nucleic acid vector for introduction into a targetbacterial host cell for expression of Type I Cas3 and Cascade proteinsin the target bacterial host cell, the vector comprising a firstnucleotide sequence encoding a Type I Cas3 and a second nucleotidesequence encoding one or more cognate Cascade proteins, wherein thefirst nucleotide sequence is under the control of a promoter forcontrolling the expression of Type I Cas3 in the target bacterial hostcell, wherein the promoter has a strength that is weaker than theAnderson Score strength of promoter BBa_J23108, wherein the targetbacterial host cell is selected from the group consisting ofFusobacteria, Bacteroides, Staphylococcus, Clostridium, Lactobacillus,Bacillus, Escherichia, Streptococcus, Streptomyces, Pseudomonas, andKlebsiella, wherein the nucleic acid vector further comprises: (i) aCRISPR array for producing crRNAs in the target bacterial host cell; or(ii) one or more nucleotide sequences encoding one or more guide RNAs(gRNA), wherein the crRNAs or gRNAs each comprise a spacer sequencecomplementary to a target sequence of the target bacterial host cell,and wherein the production strain bacterial cell does not comprise acrRNA or gRNA operable with the Cas3 to target and cut a chromosomalsequence of the production strain cell.
 2. The production strainbacterial cell of claim 1, wherein the nucleic acid vector comprises anoperon for expression of the Type I Cas3 and Cascade proteins, and: (a)the first nucleotide sequence is between the promoter and the secondnucleotide sequence in the operon; (b) the operon comprises noCas-encoding nucleotide sequences between the promoter and the firstnucleotide sequence; or (c) the operon comprises, in 5′ to 3′ direction,the promoter, the first nucleotide sequence, and the second nucleotidesequence.
 3. The production strain bacterial cell of claim 1, whereinthe promoter is a constitutive promoter.
 4. The production strainbacterial cell of claim 1, wherein the promoter is repressible.
 5. Theproduction strain bacterial cell of claim 1, wherein the promoter has astrength that is greater than the Anderson Score strength of promoterBBa_J23114.
 6. The production strain bacterial cell of claim 1, furthercomprising an origin of replication that is operable in the targetbacterial host cell.
 7. The production strain bacterial cell of claim 1,wherein the nucleic acid vector is devoid of a Cas adaption module. 8.The production strain bacterial cell of claim 1, wherein the nucleicacid vector is devoid of a nucleotide sequence encoding one or more of aCas1, Cas2, Cas4, Cas6, Cas7, and Cas8.
 9. The production strainbacterial cell of claim 1, wherein the second nucleotide sequenceencodes one or more of (a)-(g): (a) Cas11, Cas7, and Cas8a1; (b) Cas8b1,Cas7, and Cas5; (c) Cas5, Cas8c, and Cas7; (d) Cas8U2, Cas7, Cas5, andCas6; (e) Cas10d, Cas7, and Cas5; (f) Cas8e, Cas11, Cas7, Cas5, andCas6; and (g) Cas8f, Cas5, Cas7, and Cas6f.
 10. The production strainbacterial cell of claim 9, wherein the Type I Cas3 is a Cas3′ or Cas3″.11. The production strain bacterial cell of claim 9, wherein the Type ICas3 is a Cas3, Cas3′ or Cas3″, and wherein the Type I Cas3 is betweenthe promoter and the second nucleotide sequence.
 12. The productionstrain bacterial cell of claim 11, wherein the nucleic acid vector isdevoid of a nucleotide sequence encoding a further Cas between thepromoter and the Type I Cas3.
 13. The production strain bacterial cellof claim 9, wherein the vector comprises the CRISPR array, the CRISPRarray is cognate with the Type I Cas3, and wherein: (a) the CRISPR arrayis a Type IA array and the nucleic acid vector comprises Cas11, Cas7,and Cas8a1; (b) the CRISPR array is a Type IB array and the nucleic acidvector comprises Cas8b1, Cas7, and Cas5; (c) the CRISPR array is a TypeIC array and the nucleic acid vector comprises Cas5, Cas8c, and Cas7;(d) the CRISPR array is a Type IU array and the nucleic acid vectorcomprises Cas8U2, Cas7, Cas5, and Cas6; (e) the CRISPR array is a TypeID array and the nucleic acid vector comprises Cas10d, Cas7, and Cas5;(f) the CRISPR array is a Type IE array and the nucleic acid vectorcomprises Cas8e, Cas11, Cas7, Cas5, and Cas6; or (g) the CRISPR array isa Type IF array and the nucleic acid vector comprises Cas8f, Cas5, Cas7,and Cas6f.
 14. The production strain bacterial cell of claim 1, whereinthe Type I Cas3 and Cascade are: (a) Type IA Cas and Cascade proteins;(b) Type IB Cas and Cascade proteins; (c) Type IC Cas and Cascadeproteins; (d) Type ID Cas and Cascade proteins; (e) Type IE Cas andCascade proteins; (f) Type IF Cas and Cascade proteins; or (g) Type IUCas and Cascade proteins.
 15. The production strain bacterial cell ofclaim 1, wherein the Type I Cas3 and Cascade are E. coli Cas and Cascadeproteins.
 16. The production strain bacterial cell of claim 1, whereinthe promoter is operable in a target host cell selected from: anESBL-producing E. coli or E. coli ST131-O25b:H4; C. difficile resistantto one or more antibiotics selected from aminoglycosides, lincomycin,tetracyclines, erythromycin, clindamycin, penicillins, cephalosporinsand fluoroquinolones; P. aeruginosa resistant to one or more antibioticsselected from carbapenems, aminoglycosides, cefepime, ceftazidime,fluoroquinolones, piperacillin and tazobactam; carbapenem-resistantKlebsiella pneumonia; and an Extended-Spectrum Beta-Lactamase(ESBL)-producing K. pneumoniae cell.
 17. The production strain bacterialcell of claim 16, wherein the Type I Cas3 and Cascade are E. coli, C.difficile, P. aeruginosa, K. pneumoniae, P. furiosus, or B. haloduransCas and Cascade proteins.
 18. The production strain bacterial cell ofclaim 1, wherein the Type I Cas3 and Cascade are E. coli, C. difficile,P. aeruginosa, K. pneumoniae, P. furiosus, or B. halodurans Cas andCascade proteins.
 19. The production strain bacterial cell of claim 1,wherein the Type I Cas3 is a Cas3 of a CRISPR/Cas locus of E. coli, andwherein the distance between the Cas3-encoding sequence of the locus andits cognate promoter in E. coli is further than the distance between theCas3-encoding sequence and the promoter for controlling the expressionof Type I Cas3 in the nucleic acid vector.
 20. The production strainbacterial cell of claim 1, wherein the CRISPR array or the gRNA-encodingsequence(s) are under the control of a second promoter that is differentfrom the promoter that controls the expression of the Type I Cas3. 21.The production strain bacterial cell of claim 1, wherein the nucleicacid vector is a plasmid or phagemid.
 22. The production strainbacterial cell of claim 1, wherein the production strain bacterial cellcomprises a nucleotide sequence whose expression is inducible to producephage coat proteins in the cell of the production strain, wherein theproduction strain bacterial cell comprises amplified copies of thenucleic acid vector, wherein the production strain bacterial cell iscapable of packaging the amplified copies of the nucleic acid vectorinto phage particles or non-self-replicative transduction particles forintroducing the amplified copies of the nucleic acid vector into thetarget host cell.
 23. The production strain bacterial cell of claim 22,wherein the nucleic acid vector is a plasmid or phagemid and thedelivery vehicle is a non-replicative transduction particle.
 24. Theproduction strain bacterial cell of claim 1, wherein the secondnucleotide sequence is under the control of the same promoter as thefirst nucleotide sequence.
 25. The production strain bacterial cell ofclaim 1, wherein the target sequence of the target bacterial host cellis a chromosomal sequence of the target bacterial host cell.
 26. Theproduction strain bacterial cell of claim 1, wherein the productionstrain bacterial cell is an Escherichia coli (E. coli) cell.